Alteration of tobacco alkaloid content through modification of specific cytochrome p450 genes

ABSTRACT

Compositions and methods for reducing the level of nornicotine and N′-nitrosonornicotine (NNN) in  Nicotiana  plants and plant parts thereof are provided. The compositions comprise isolated polynucleotides and polypeptides for cytochrome P450 s  that are involved in the metabolic conversion of nicotine to nornicotine in these plants. Expression cassettes, vectors, plants, and plant parts thereof comprising inhibitory sequences that target expression or function of the disclosed cytochrome P450 polypeptides are also provided. Methods for the use of these novel sequences to inhibit expression or function of cytochrome P450 polypeptides involved in this metabolic conversion are also provided. The methods find use in the production of tobacco products that have reduced levels of nornicotine and its carcinogenic metabolite, NNN, and thus reduced carcinogenic potential for individuals consuming these tobacco products or exposed to secondary smoke derived from these products.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.16/860,542, filed Apr. 28, 2020, which is a continuation of U.S.application Ser. No. 15/631,873, filed Jun. 23, 2017, which is acontinuation of U.S. application Ser. No. 14/950,155, filed Nov. 24,2015, which is a continuation of U.S. application Ser. No. 13/361,235,filed Jan. 30, 2012, which is a divisional of U.S. application Ser. No.12/269,531, filed Nov. 12, 2008, which claims the benefit of U.S.Provisional Application No. 60/987,243, filed Nov. 12, 2007, thecontents of each of which are hereby incorporated in their entirety byreference herein.

U.S. application Ser. No. 14/950,155, filed Nov. 24, 2015, is also acontinuation of U.S. application Ser. No. 13/361,159, filed Jan. 30,2012, which is a divisional of U.S. application Ser. No. 12/269,531,filed Nov. 12, 2008, which claims the benefit of U.S. ProvisionalApplication No.: 60/987,243, filed Nov. 12, 2007, the contents of eachof which are hereby incorporated in their entirety by reference herein.

REFERENCE TO SEQUENCE LISTING SUBMITTED ELECTRONICALLY

An official copy of the Sequence Listing is submitted electronically viaEFS-Web as an ASCII formatted Sequence Listing with a file named“575405SEQLIST.txt,” created on May 18, 2022, having a size of 136 KB.The Sequence Listing contained in this ASCII formatted document is partof the specification and is herein incorporated by reference in itsentirety.

FIELD OF THE INVENTION

The present invention relates to compositions and methods for reducingthe level of nornicotine and its metabolite, N′-nitrosonornicotine(NNN), in a plant that is a member of the genus Nicotiana, particularlycompositions and methods for inhibiting expression or function of acytochrome P450 polypeptide involved in the metabolic conversion ofnicotine to nornicotine.

BACKGROUND OF THE INVENTION

A predominant alkaloid found in commercial tobacco varieties isnicotine, typically accounting for 90%-95% of the total alkaloid pool.The remaining alkaloid fraction is primarily three additional pyridinealkaloids: nornicotine, anabasine and anatabine. Nornicotine isgenerated directly from nicotine by nicotine N-demethylase. Nornicotineusually represents less than 5% of the total pyridine alkaloid pool.However, tobacco plants that initially produce very low amounts ofnornicotine can give rise to progeny that metabolically “convert” alarge percentage of leaf nicotine to nornicotine. This process is termed“conversion.” In tobacco plants that have genetically converted (i.e.,“converters”), the great majority of nornicotine production occursduring senescence and curing of a mature leaf (Wernsman & Matzinger(1968) Tob. Sci. 12:226-228). Burley tobaccos are particularly prone togenetic conversion, with rates as high as 20% per generation observed insome cultivars.

During curing and processing of the tobacco leaf, a portion of thenornicotine is metabolized to NNN, a tobacco-specific nitrosamine (TSNA)alleged to be carcinogenic in laboratory animals (Hecht & Hoffmann(1990) Cancer Surveys 8:273-294; and Hoffmann et al. (1994) J. Toxicol.Environ. Health 41:1-52; Hecht (1998) Chem. Res. Toxicol. 11:559-603).In flue-cured tobaccos, TSNAs predominantly form through a reaction ofalkaloids with minute amounts of nitrogen oxides present in combustiongases in a direct-fired heating systems used in traditional curing barns(Peele & Gentry (1999) “Formation of tobacco-specific nitrosamines influe-cured tobacco,” CORESTA Meeting, Agro-Phyto Groups, Suzhou, China).The combustion gases, however, can be eliminated when curing barns areretrofitted with heat-exchangers, which eliminate the mixing ofcombustion gases with curing air, thereby reducing TSNAs in tobaccoscured in this manner (Boyette & Hamm (2001) Rec. Adv. Tob. Sci.27:17-22.). In contrast, in air-cured Burley tobaccos, TSNA formationprimarily proceeds through a reaction of tobacco alkaloids with nitrite,a process catalyzed by leaf-borne microbes (Bush et al. (2001) Rec. Adv.Tob. Sci. 27:23-46). Thus far, attempts to reduce TSNAs throughmodification of curing conditions while maintaining acceptable qualitystandards have not been successful for air-cured tobaccos.

In Burley tobacco plants, a positive correlation exists between thenornicotine content of a leaf and an amount of NNN that accumulates inthe cured leaf (Bush et al. (2001) Rec. Adv. Tob. Sci, 27:23-46; and Shiet al. (2000) Tob. Chem. Res. Conf. 54:Abstract 27). However, keepingnornicotine levels at a minimum is difficult in Burley tobacco plantsbecause of conversion. Plant breeders and seed producers aretraditionally responsible for minimizing the number of Burley tobaccoplants that accumulate high levels of nornicotine. Though the percentageof converters that are ultimately grown in fields are reduced throughroguing converters during propagation of seed stocks. Unfortunately,this process is costly, time-consuming and imperfect.

Once a plant converts, the high nornicotine trait is inherited as asingle dominant gene (Griffith et al. (1955) Science 121:343-344; Burke& Jeffrey (1958) Tob. Sci. 2:139-141; and Man et al. (1964) Crop Sci.4:349-353). The nature of this gene, however, is currently unknown. Inthe simplest of scenarios, the conversion locus may represent anonfunctional nicotine N-demethylase gene that regains its function inconverters, possibly through the mobilization of a mutation-inductingtransposable element. Alternatively, the converter locus may encode aprotein that initiates a cascade of events that ultimately enablesconverters to metabolize nicotine to nornicotine, meaning that multiplegenes may be involved.

Regardless of whether there are one or many genes associated withconversion, the gene(s) encoding polypeptides having nicotinedemethylase activity play a pivotal role in this process. Although theinability to purify active nicotine N-demethylase from crude extractshas impeded the isolation and identification of this enzyme, there issome evidence that a member of the cytochrome P450 superfamily ofmonooxygenases may be involved (Hao & Yeoman (1996) Phytochem.41:477-482; Hao & Yeoman (1996) Phytochem. 42:325-329; Chelvarajan etal. (1993) J. Agric, Food Chem. 41:858-862; and Hao & Yeoman (1998) J.Plant Physiol. 152:420-426). Unfortunately, these studies are notconclusive, as classic P450 inhibitors, such as carbon monoxide andtetcylasis, fail to lower enzyme activity at rates comparable to otherreported P450-mediated reactions (Chelvarajan et al. (1993) J. Agric.Food Chem. 41:858-862).

Furthermore, cytochrome P450s are ubiquitous, transmembrane proteinsthat participate in metabolizing a wide range of compounds (reviewed bySchuler (1996) Crit. Rev. Plant Sci. 15:235-284; and Schuler &Werck-Reichhart (2003) Annu. Rev. Plant Biol. 54:629-667). Examples ofbiochemical reactions mediated by cytochrome P450s includehydroxylations, demethylations and epoxidations. In plants, cytochromeP450 gene families are very large. For example, total genome sequenceexamination revealed 272 predicted cytochrome P450 genes in Arabidopsisand at least 455 unique cytochrome P450 genes in rice (see, e.g., Nelsonet al. (2004) Plant Physiol. 135(2):756-772). Even though cytochromeP450s have been implicated in the conversion of nicotine to nornicotine,identification of key participating members of this protein familyremains a challenge.

Aside from serving as a precursor for NNN, recent studies suggest thatthe nornicotine found in tobacco products has undesirable healthconsequences. For example, Dickerson & Janda demonstrated thatnornicotine causes aberrant protein glycosylation within a cell(Dickerson & Janda (2002) Proc. Natl. Acad. Sci USA 99:15084-15088).Likewise, concentrations of nornicotine-modified proteins were muchhigher in plasma of smokers compared to nonsmokers. Furthermore,nornicotine can covalently modify commonly prescribed steroid drugs suchas prednisone, which can alter both the efficacy and toxicity of thesedrugs.

In view of the difficulties associated with conversion, as well as theundesirable health effects of nornicotine accumulation, improved methodsfor reducing the nornicotine content in tobacco varieties, particularlyBurley tobacco plants, are therefore desirable. Such methods would notonly help ameliorate the potential negative health consequences of thenornicotine per se as described above, but also help to reduce NNNlevels.

SUMMARY OF THE INVENTION

Compositions and methods are provided for reducing the nornicotinecontent in plants that are members of the genus Nicotiana. Compositionsinclude isolated cytochrome P450 polynucleotides and polypeptides thatare involved in conversion of nicotine to nornicotine in plants,particularly Nicotiana species. Isolated polynucleotides include thosethat comprise a nucleic acid sequence as set forth in SEQ ID NO:1, 3 or4, a nucleic acid sequence encoding a polypeptide as set forth in SEQ IDNO:2, 5-12, 14-24, and fragments and variants thereof. Isolatedpolypeptides of the invention include those that comprise an amino acidsequence as set forth in SEQ ID NO:2, 5-12 or 14-24, an amino acidsequence encoded by the nucleic acid sequence set forth in SEQ ID NO:1,3 or 4, and fragments and variants thereof.

In a first aspect, the present invention is summarized aspolynucleotides that can suppress expression of a nicotine demethylaseinvolved in the metabolic conversion of nicotine to nornicotine in aplant, including the nicotine demethylases of the present invention. Thepresent invention provides an isolated polynucleotide having a promotercapable of functioning in a plant cell operably linked to a nucleic acidsequence comprising a region of between about 100 nucleic acids andabout 350 nucleic acids of SEQ ID NO:1 obtained from a sequence selectedfrom the group of nucleic acids at position 253, 353, 647, 733, 1050,1397 and combinations thereof. The present invention also provides anisolated polynucleotide comprising a nucleic acid sequence encoding agreen-leaf nicotine demethylase, where the amino acid sequence of theencoded nicotine demethylase has a substitution at an amino acid residuein a position selected from the group consisting of residues 235, 449,174, 410, 224, 72, 143 and 422, where the numbering is according to SEQID NO:2. Also provided is an isolated polynucleotide comprising apromoter capable of functioning in a plant cell operably linked to anucleic acid sequence comprising a region of between about 100 nucleicacids and about 350 nucleic acids of SEQ ID NO:4. Further provided is anisolated polynucleotide encoding an amino acid sequence as set forth inSEQ ID NO:2 with a mutation of a residue that differs from the otherP450 polypeptides to a conserved residue. Alternatively provided is anisolated polynucleotide encoding an amino acid sequence as set forth inSEQ ID NO:2 with a mutation at a position selected from the groupconsisting of residues 85, 118, 216, 245 and 466, where the residue at85 is not an isoleucine, the residue at 118 is not an asparagine, theresidue at 216 is not tyrosine, the residue at 245 is not a tyrosine,and the residue at 466 is not valine.

In a second aspect, the present invention is summarized as an expressioncassette comprising a polynucleotide encoding an amino acid sequence ofSEQ ID NO:2 operably linked to a promoter that is functional in a plantcell. The present invention provides an expression cassette comprises apolynucleotide comprising a nucleic acid sequence of SEQ ID NO:3, or afragment of at least 25 contiguous nucleic acids thereof, operablylinked to a promoter that is functional in a plant cell. Also providedis an isolated polynucleotide comprising at least 25 nucleotides of anucleic acid sequence of SEQ ID NO:3. Further provided is an isolatedpolypeptide comprising an amino acid sequence selected from the groupconsisting of SEQ ID NO:2, 5-12 and 14-24. In one embodiment of thesecond aspect, the isolated polypeptide comprises an amino acid sequence99% identical to SEQ ID NO:2, such that it is capable of convertingnicotine to nornicotine in green leaves of tobacco.

In a third aspect, the present invention is summarized as a plant of thegenus Nicotiana or a plant part thereof comprising an expressioncassette, the cassette encoding SEQ ID NO:2, a fragment thereof, or acomplement of either. Also provided is a transgenic Nicotiana planthaving a lower level of nicotine to nornicotine conversion rate in greenleaves compared to a non-transgenic plant, the plant comprising anexogenous nucleic acid construct comprising a promoter capable offunctioning in a plant cell operably linked to a polynucleotide having afirst nucleic acid sequence comprising a region of between about 100nucleic acids and about 350 nucleic acids of a green-leaf tobacconicotine demethylase sequence encoding an amino acid sequence of SEQ IDNO:2 and a second nucleic acid sequence capable of forming a doublestranded RNA with the first sequence. Further provided is a transgenicNicotiana plant having a lower level of nicotine to nornicotineconversion rate in green leaves compared to a non-transgenic plant, theplant comprising an exogenous nucleic acid construct comprising apromoter capable of functioning in a plant cell operably linked to apolynucleotide having a first nucleic acid sequence comprising a regionof between about 100 nucleic acids and about 350 nucleic acids of agreen-leaf tobacco nicotine demethylase sequence having the nucleic acidsequence of SEQ ID NO:3 and a second nucleic acid sequence capable offorming a double stranded RNA with the first sequence is provided. Alsoprovided is a transgenic Nicotiana plant having a lower level ofnicotine to nornicotine conversion rate in green leaves compared to anon-transgenic plant, the plant comprising an exogenous nucleic acidconstruct comprising a promoter capable of functioning in a plant celloperably linked to a polynucleotide having a first nucleic acid sequencecomprising a region of between about 100 nucleic acids and about 350nucleic acids of a green-leaf tobacco nicotine demethylase sequencehaving a nucleic acid sequence of SEQ ID NO:4 and a second nucleic acidsequence capable of forming a double stranded RNA with the firstsequence.

In a fourth aspect, the present invention is summarized as a seed of atransgenic Nicotiana plant having a lower level of nicotine tonornicotine conversion rate in green leaves compared to a non-transgenicplant, the plant comprising a heterologous promoter capable offunctioning in a plant cell operably linked to a polynucleotide having afirst nucleic acid sequence comprising a region of between about 100nucleic acids and about 350 nucleic acids of a green-leaf tobacconicotine demethylase sequence having an amino acid sequence of SEQ IDNO:4 and a second nucleic acid sequence capable of forming a doublestranded RNA with the first sequence. Also provided is a tissue cultureof regenerable tobacco cells comprising a plant cell that comprises afirst polynucleotide having a fragment of the nucleic acid sequence ofSEQ ID NO:1, 3 or 4 and a second polynucleotide capable of forming adouble stranded RNA with the first.

In a fifth aspect, the present invention is summarized as a tobaccoproduct comprising a transgenic Nicotiana plant cell having a lowerlevel of nicotine to nornicotine conversion rate in green leavescompared to a non-transgenic plant, the plant cell comprising aheterologous promoter capable of functioning in a plant cell operablylinked to a polynucleotide having a first nucleic acid sequencecomprising a region of between about 100 nucleic acids and about 350nucleic acids of a green-leaf tobacco nicotine demethylase sequencehaving a nucleic acid sequence of SEQ ID NO:4 and a secondpolynucleotide capable of forming a double stranded RNA with the first.Also provided is a tobacco cell having a genome altered to inhibit theexpression of at least a green-leaf nicotine demethylase, where the cellis homozygous for a mutation in the gene encoding the green-leafnicotine demethylase. Further provided is a tobacco cell comprising atransgene containing green-leaf nicotine demethylase nucleic acidsequence that flanks a selectable marker gene, where the selectablemarker gene disrupts the nicotine demethylase gene, thereby producing atobacco cell where the endogenous green-leaf nicotine demethylase genehas been disrupted. Alternatively provided is a tobacco cell comprisinga transgene containing green-leaf nicotine demethylase nucleic acidsequence that flanks a selectable marker gene, where the selectablemarker gene disrupts the nicotine demethylase gene, thereby producing atobacco cell where the endogenous green-leaf nicotine demethylase genehas been disrupted.

In a sixth aspect, the present invention is summarized as a method forreducing nornicotine levels in a plant part derived from a plant of thegenus Nicotiana, the method comprising a) inhibiting expression of anicotine demethylase, where the nicotine demethylase has an amino acidsequence set forth in the group consisting of SEQ ID NOs: 2 and 5-12;and b) reducing nornicotine levels in a plant part derived from a plantof the genus Nicotiana. Also provided is a method for reducingnornicotine levels in a tobacco product, the method comprising a)growing a transgenic tobacco plant, where the plant having a plant partthat comprises an expression cassette comprising a heterologous promoterand a nicotine demethylase, where the nicotine demethylase has an aminoacid sequence selected from the group consisting of SEQ ID NOs:2 and5-12; and b) preparing a tobacco product from the tobacco plant part.Further provided is a method for reducing nornicotine levels in atobacco product, the method comprising a) growing a transgenic tobaccoplant, where the plant has a plant part that comprises an antibody thatspecifically binds a polypeptide with an amino acid sequence selectedfrom the group consisting of SEQ ID NOs:2 and 5-12; and b) preparing atobacco product from the tobacco plant part. Alternatively provided is amethod for reducing nornicotine levels in a tobacco product, the methodcomprising a) growing a transgenic tobacco plant, where the plant has aplant part that comprises a fragment of a green-leaf nicotinedemethylase operably linked to a heterologous promoter and the fragmenthas at least 25 contiguous nucleic acids from a polynucleotide encodinga polypeptide selected from the group consisting of SEQ ID NOs:2 and5-12; and b) preparing a tobacco product from the tobacco plant part.Also provided is a method for reducing nornicotine levels in a plantpart derived from a plant of the genus Nicotiana, the method comprisinga) modifying the functional CYP82E5v2 allele to change alleles to anonfunctional CYP82E5v2, where the nonfunctional CYP82E5v2 has asubstitution at an amino acid residue in a position selected from thegroup consisting of residues 235, 449, 174, 410, 224, 72, 143 and 422,where the numbering is according to SEQ ID NO:2; and b) reducing thelevel of nornicotine in a plant part derived from a plant of the genusNicotiana. The present invention also provides a method for reducing thecarcinogenic potential of a tobacco product, the method comprisingpreparing the tobacco product by a) growing a transgenic tobacco plant,where the plant comprises a plant part that comprises a fragment of agreen-leaf nicotine demethylase operably linked to a heterologouspromoter and the fragment comprises at least 25 contiguous nucleic acidsfrom a polynucleotide encoding a polypeptide selected from the groupconsisting of SEQ ID NOs:2 and 5-12; and b) preparing a tobacco productfrom the tobacco plant part. Further, the present invention provides amethod of reducing the conversion of nicotine to nornicotine in aNicotiana plant comprising: a) transforming a Nicotiana plant with annucleic acid construct comprising a promoter capable of functioning in aplant cell operably linked to a polynucleotide having a first nucleicacid sequence comprising a region of between 100 nucleic acids and about350 nucleic acids of SEQ ID NO:1, 3 or 4 and a second nucleic acidsequence capable of forming a double stranded RNA with the firstsequence; and b) regenerating a transgenic Nicotiana plant.

In a seventh aspect, the present invention is summarized as a method ofscreening for a green-leaf nicotine demethylase sequence comprising a)obtaining a nucleic acid sequence that has at least 200 nucleic acids ofsequence identity with SEQ ID NO:1; and b) identifying a codon sequenceencoding for a stop codon at position 422 of an encoded polypeptide,where the numbering is according to SEQ ID NO:2. Also provided is amethod of screening for green-leaf nicotine demethylase sequencecomprising a) obtaining a nucleic acid sequence that has at least 200nucleic acids of sequence identity with SEQ ID NO:1; and b) identifyinga codon sequence encoding for a codon that is not a proline at position449 of an encoded polypeptide, where the numbering is according to SEQID NO:2. Further provided is a method for identifying a tobacco plantwith low levels of nornicotine, the method comprising a) obtaining a DNAsample from a tobacco plant of interest; and b) screening the sample fora mutation in SEQ ID NO:1. The present invention also provides a methodfor reducing the level of nornicotine in a plant part derived from aplant of the genus Nicotiana, the method comprising a) inhibitingexpression of a CYP82E4v2 nicotine demethylase and CYP82E5v2 nicotinedemethylase; and b) reducing the level of nornicotine in a plant partderived from a plant of the genus Nicotiana. Also included is tobaccoplant material comprising a polypeptide with an amino acid sequence ofSEQ ID NO:13 having a mutation at a position selected from the groupconsisting of residues 458, 364, 329 and combinations thereof. Furtherincluded is tobacco plant material comprising a CYP82E4v2 having amutation at a position selected from the group consisting of residues458, 364, 329 and combinations thereof, where the numbering correspondsto SEQ ID NO:13. Also included is tobacco plant material comprising aCYP82E4v2 having a mutation at residue 376, where the residue is notvaline and the numbering of residues corresponds to SEQ ID NO:13.

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1A-C show an amino acid sequence alignment of CYP82E2 gene familymembers that have been assayed for nicotine demethylase activity inyeast and/or transgenic plants. Sequences in italics and underlined arepositive for nicotine demethylase activity (CYP82E4v2 and CYP82E5v2);sequences titled in black failed to show activity in an assay. Residuesthat differ among the members are shaded in grey. In FIG.1, CYP82E4v2 isset forth in SEQ ID NO:13; CYP82E4v6 is set forth in SEQ ID NO:26;CYP82E4v12 is set forth in SEQ ID NO:27; 58-166 is set forth in SEQ IDNO:28; CYP82E3 is set forth in SEQ ID NO:29; CYP82E2v1 is set forth inSEQ ID NO:30; CYP82E2v2 is set forth in SEQ ID NO:31; and CYP82E5v2 isset forth in SEQ ID NO:2.

DESCRIPTION OF THE NUCLEIC ACID SEQUENCES

SEQ ID NO:1 sets forth a nucleic acid sequence of a coding region ofCYP82E5v2.

SEQ ID NO:2 sets forth an amino acid sequence of a CYP82E5v2.

SEQ ID NO:3 sets forth a nucleic acid sequence of an intron ofCYP82E5v2.

SEQ ID NO:4 sets forth a nucleic acid sequence of a genomic CYP82E5v2.

SEQ ID NO:5 sets forth an amino acid sequence of a CYP82E5v2 P235S.

SEQ ID NO:6 sets forth an amino acid sequence of a CYP82E5v2 P449L.

SEQ ID NO:7 sets forth an amino acid sequence of a CYP82E5v2 S174L.

SEQ ID NO:8 sets forth an amino acid sequence of a CYP82E5v2 A410V.

SEQ ID NO:9 sets forth an amino acid sequence of a CYP82E5v2 M224I.

SEQ ID NO:10 sets forth an amino acid sequence of a CYP82E5v2 P72L.

SEQ ID NO:11 sets forth an amino acid sequence of a CYP82E5v2 L143F.

SEQ ID NO:12 sets forth an amino acid sequence of a CYP82E5v2 W422Stop.

SEQ ID NO:13 sets forth an amino acid sequence of a CYP82E4v2.

SEQ ID NO:14 sets forth an amino acid sequence of a CYP82E4v2 P458S.

SEQ ID NO:15 sets forth an amino acid sequence of a CYP82E4v2 K364N.

SEQ ID NO:16 sets forth an amino acid sequence of a CYP82E4v2 P38L.

SEQ ID NO:17 sets forth an amino acid sequence of a CYP82E4v2 E201K.

SEQ ID NO:18 sets forth an amino acid sequence of a CYP82E4v2 R169Q.

SEQ ID NO:19 sets forth an amino acid sequence of a CYP82E4v2 G459R.

SEQ ID NO:20 sets forth an amino acid sequence of a CYP82E4v2 E296K.

SEQ ID NO:21 sets forth an amino acid sequence of a CYP82E4v2 T427I.

SEQ ID NO:22 sets forth an amino acid sequence of a CYP82E4v2 W329Stop.

SEQ ID NO:23 sets forth an amino acid sequence of a CYP82E4v2 V376M.

SEQ ID NO:24 sets forth an amino acid sequence of a CYP82E4v2 D171N.

SEQ ID NO:25 sets forth an amino acid sequence of a CYP82E4.

SEQ ID NO:26 sets forth an amino acid sequence of a CYP82E4v6.

SEQ ID NO:27 sets forth an amino acid sequence of a CYP82E4v12.

SEQ ID NO:28 sets forth an amino acid sequence of a 58-166.

SEQ ID NO:29 sets forth an amino acid sequence of a CYP82E3.

SEQ ID NO:30 sets forth an amino acid sequence of a CYP82E2v1.

SEQ ID NO:31 sets forth an amino acid sequence of a CYP82E2v2.

SEQ ID NO:32 sets forth a nucleic acid sequence of a forward primer forexon 1 of CYP82E4v2.

SEQ ID NO:33 sets forth a nucleic acid sequence of a reverse primer forexon 1 of CYP82E4v2.

SEQ ID NO:34 sets forth a nucleic acid sequence of a forward primer forexon 2 of CYP82E4v2.

SEQ ID NO:35 sets forth a nucleic acid sequence of a reverse primer forexon 2 of CYP82E4v2.

SEQ ID NO:36 sets forth a nucleic acid sequence of a forward primer forexon 1 of CYP82E5v2.

SEQ ID NO:37 sets forth a nucleic acid sequence of a reverse primer forexon 1 of CYP82E5v2.

SEQ ID NO:38 sets forth a nucleic acid sequence of a forward primer forexon 2 of CYP82E5v2.

SEQ ID NO:39 sets forth a nucleic acid sequence of a reverse primer forexon 2 of CYP82E5v2.

SEQ ID NO:40 sets forth a nucleic acid sequence of a primer E5Gen_F1.

SEQ ID NO:41 sets forth a nucleic acid sequence of a primer E5Gen_R1.

SEQ ID NO:42 sets forth a nucleic acid sequence of a primer E5Gen_F2.

SEQ ID NO:43 sets forth a nucleic acid sequence of a primer E5Gen_R2.

SEQ ID NO:44 sets forth a nucleic acid sequence of a primer E4Rt_F.

SEQ ID NO:45 sets forth a nucleic acid sequence of a primer E4Rt_R.

SEQ ID NO:46 sets forth a nucleic acid sequence of a primer E5Rt_F.

SEQ ID NO:47 sets forth a nucleic acid sequence of a primer E5Rt_R.

SEQ ID NO:48 sets forth a nucleic acid sequence of a primer G3PDH_F.

SEQ ID NO:49 sets forth a nucleic acid sequence of a primer G3PDH_R.

SEQ ID NO:50 sets forth a nucleic acid sequence of a coding region ofCYP82E4v2 and the encoded protein (i.e., SEQ ID NO:13).

DEFINITIONS

The present invention includes compositions and methods for inhibitingexpression or function of nicotine demethylase polypeptides that areinvolved in the metabolic conversion of nicotine to nornicotine in aplant, particularly plants of the Nicotiana genus, including tobaccoplants of various commercial varieties.

As used herein, “inhibit,” “inhibition” and “inhibiting” are defined asany method known in the art or described herein, which decreases theexpression or function of a gene product of interest (i.e., the targetgene product).

“Inhibiting” can be in the context of a comparison between two plants,for example, a genetically altered plant versus a wild-type plant. Thecomparison can be between plants, one of which lacks a DNA sequencecapable of reducing the agent. Inhibition of expression or function of atarget gene product also can be in the context of a comparison betweenplant cells, organelles, organs, tissues or plant parts within the sameplant or between different plants, and includes comparisons betweendevelopmental or temporal stages within the same plant or plant part orbetween plants or plant parts.

“Inhibiting” can include any relative decrement of function orproduction of a gene product of interest, up to and including completeelimination of function or production of that gene product. When levelsof an agent are compared, such a comparison is preferably carried outbetween organisms with a similar genetic background. Preferably, asimilar genetic background is a background where the organisms beingcompared share 50% or greater, more preferably 75% or greater, and, evenmore preferably 90% or greater sequence identity of nuclear geneticmaterial. A similar genetic background is a background where theorganisms being compared are plants, and the plants are isogenic exceptfor any genetic material originally introduced using planttransformation techniques or a mutation generated by human intervention.Measurement of the level or amount of an agent may be carried out by anysuitable method, non-limiting examples of which include, but are notlimited to, comparison of mRNA transcript levels, protein or peptidelevels, and/or phenotype, especially the conversion of nicotine tonornicotine. As used herein, mRNA transcripts can include processed andnon-processed mRNA transcripts, and polypeptides or peptides can includepolypeptides or peptides with or without any post-translationalmodification.

As used herein, “host cell” means a cell that comprises a heterologousnucleic acid sequence of the invention. Though the nucleic acidsequences of the invention, and fragments and variants thereof, can beintroduced into any cell of interest, of particular interest are plantcells, more particularly cells of a Nicotiana plant species, forexample, the tobacco plant species and varieties described herein below.

As used herein, “variant” means a substantially similar sequence. Avariant can have different function or a substantially similar functionas a wild-type polypeptide of interest. For a nicotine demethylase, asubstantially similar function is at least 99%, 98%, 97%, 95%, 90%, 85%,80%, 75%, 60%, 50%, 25% or 15% of wild-type enzyme function ofconverting nicotine to nornicotine under the same conditions or in anear-isogenic line. A wild-type CYP82E5v2 is SEQ ID NO:2. A wild-typeCYP82E4v2 is SEQ ID NO:13. As used herein, a “variant polynucleotide” or“variant polypeptide” means a nucleic acid or amino acid sequence thatis not wild-type.

A variant can have one addition, deletion or substitution; two or lessadditions, deletions or substitutions; three or less additions,deletions or substitutions; four or less additions, deletions orsubstitutions; or five or less additions, deletions or substitutions. Amutation includes additions, deletions, and substitutions. Suchdeletions or additions can be at the C-terminus, N-terminus or both theC- and N-termini. Fusion polypeptides or epitope-tagged polypeptides arealso included in the present invention. “Silent” nucleotide mutations donot change the encoded amino acid at a given position. Amino acidsubstitutions can be conservative. A conservative substitution is achange in the amino acid where the change is to an amino acid within thesame family of amino acids as the original amino acid. The family isdefined by the side chain of the individual amino acids. A family ofamino acids can have basic, acidic, uncharged polar or nonpolar sidechains. See, Alberts et al., (1994) Molecular biology of the cell (3rded., pages 56-57, Garland Publishing Inc., New York, N.Y.), incorporatedherein by reference as if set forth in its entirety. A deletion,substitution or addition can be to the amino acid of another CYP82Efamily member in that same position. See, FIG. 1A-C. As used herein, a“fragment” means a portion of a polynucleotide or a portion of apolypeptide and hence protein encoded thereby.

As used herein, “plant part” means plant cells, plant protoplasts, plantcell tissue cultures from which a whole plant can be regenerated, plantcalli, plant clumps and plant cells that are intact in plants or partsof plants such as embryos, pollen, anthers, ovules, seeds, leaves,flowers, stems, branches, fruit, roots, root tips and the like. Progeny,variants and mutants of regenerated plants are also included within thescope of the present invention, provided that they comprise theintroduced polynucleotides of the invention. As used herein, “tobaccoplant material” means any portion of a plant part or any combination ofplant parts.

As used herein, “operably linked” means a functional linkage between twoor more elements. For example, an operable linkage between apolynucleotide of interest and a regulatory sequence (i.e., a promoter)is a functional link that allows for expression of the polynucleotide ofinterest. Operably linked elements may be contiguous or non-contiguous.When used to refer to the fusing of two protein coding regions, byoperably linked is intended that the coding regions are in the samereading frame.

As used herein, “heterologous” means a sequence that originates from aforeign species, or, if from the same species, is substantially modifiedfrom its native form in composition and/or genomic locus by deliberatehuman intervention. For example, a promoter operably linked to aheterologous polynucleotide is from a species different from the speciesfrom which the polynucleotide was derived, or, if from thesame/analogous species, one or both are substantially modified fromtheir original form and/or genomic locus, or the promoter is not thenative promoter for the operably linked polynucleotide. Furthermore, asused herein, “chimeric gene” means a coding sequence operably linked toa transcription initiation region that is heterologous to the codingsequence.

DETAILED DESCRIPTION OF THE INVENTION

Nicotine Demethylase Polynucleotides and Polypeptides, and Variants andFragments Thereof

Compositions of the present invention include cytochrome P450polypeptides. Cytochrome P450 polypeptides can have nicotine demethylaseactivity. Such nicotine demethylase polynucleotides and polypeptides areinvolved in the metabolic conversion of nicotine to nornicotine inplants, including commercial varieties of tobacco plants. Also includedare variants of such nicotine demethylases. In particular, compositionsof the invention include isolated polypeptides comprising amino acidsequences as shown in

SEQ ID NOS:2 and 5-24, isolated polynucleotides comprising the nucleicacid sequences as shown in SEQ ID NOS:1, 3 and 4, and the isolatedpolynucleotides encoding polypeptides comprising amino acid sequences ofSEQ ID NOS:2 and 5-24. The polynucleotides of the present invention canfind use in inhibiting expression of nicotine demethylase polypeptidesor variants thereof that are involved in the metabolic conversion ofnicotine to nornicotine in plants, particularly tobacco plants. Some ofthe polynucleotides of the invention have mutations that inhibitnicotine demethylase activity of the wild-type nicotine demethylase. Theinhibition of polypeptides of the present invention is effective inlowering nornicotine levels in tobacco lines where genetic conversionoccurs in less than 30%, 50%, 70% and 90% of the population, such asflue-cured tobaccos. The inhibition of polypeptides of the presentinvention is effective in lowering nornicotine levels in tobaccopopulations where genetic conversion occurs in at least 90%, 80%, 70%,60% and 50% of a plant population. A population preferably containsgreater than about 25, 50, 100, 500, 1,000, 5,000 or 25,000 plantswhere, more preferably at least about 10%, 25%, 50%, 75%, 95% or 100% ofthe plants comprise a polypeptide of the present invention.

The present invention further provides expression cassettes comprisingall or a portion of the polynucleotides having a nucleic acid sequenceset forth in SEQ ID NO:1, 3 or 4, and the isolated polynucleotidesencoding polypeptides having an amino acid sequence of SEQ ID NOS: 2 and5-24, a complement or fragment thereof, or a sequence having substantialsequence identity to SEQ ID NO:1, 3 or 4, or the polynucleotidesencoding polypeptides having an amino acid sequence of SEQ ID NOS:2 and5-24, or a complement or fragment thereof, operably linked to aheterologous promoter that is functional in a plant cell for use inexpressing an inhibitory RNA transcript that interferes with expression(i.e., transcription and/or translation) of nicotine demethylasepolypeptides. In some embodiments, the expression cassettes comprise thenucleotide sequence as shown in SEQ ID NO:1, 3 or 4, a complement orfragment thereof, or a sequence having substantial sequence identity toSEQ ID NO:1, 3 or 4, or a complement or fragment thereof. Introductionof these expression cassettes into a Nicotiana plant of interest;particularly a tobacco plant of varieties commonly known as flue orbright varieties, Burley varieties, dark varieties and oriental/Turkishvarieties, results in the production of tobacco plants having reducedamounts of nornicotine and NNN. Leaf and stem material from thesetransgenic plants can be used to produce a variety of tobacco productshaving, reduced levels of nornicotine, and a concomitant reduction NNN.

The nicotine demethylase polynucleotides and encoded polypeptides of thepresent invention include a novel cytochrome P450 gene, designated theCYP82E5v2 nicotine demethylase gene, that is newly identified as havinga role in the metabolic conversion of nicotine to nornicotine in tobaccoplants. Suppression of the expression of the encoded polypeptide intransgenic tobacco plants results in a significant reduction in theaccumulation of nornicotine in the green leaves of these transgenicplants. While not being bound by theory, the metabolic role of thesepolypeptides may be a direct one, i.e., directly catalyzing theN-demethylation reaction, or an indirect one, i.e., in the form ofproduction of a product that leads to the up-regulation of the nicotinedemethylase activity of the leaf. Regardless of the mechanism, any meansby which expression and/or function of the polypeptides of the presentinvention are targeted for inhibition or site-directed mutagenesiswithin a Nicotiana plant will be effective in reducing nornicotinelevels, and levels of NNN, within leaves and stems of these plants.

The invention encompasses isolated or substantially purifiedpolynucleotide or polypeptide compositions of the present invention. An“isolated” or “purified” polynucleotide or polypeptide, or biologicallyactive portion thereof, is substantially or essentially free fromcomponents that normally accompany or interact with the polynucleotideor protein as found in its naturally occurring environment. Thus, anisolated or purified polynucleotide or polypeptide is substantially freeof other cellular material or culture medium when produced byrecombinant techniques, or substantially free of chemical precursors orother chemicals when chemically synthesized. Optimally, an “isolated”polynucleotide is free of sequences (optimally protein encodingsequences) that naturally flank the polynucleotide (i.e., sequenceslocated at the 5′ and 3′ ends of the polynucleotide) in the genomic DNAof the organism from which the polynucleotide is derived. For example,in various embodiments, the isolated polynucleotide can contain lessthan about 5 kb, 4 kb, 3 kb, 2 kb, 1 kb, 0.5 kb or 0.1 kb of nucleotidesequence that naturally flank the polynucleotide in genomic DNA of thecell from which the polynucleotide is derived. A polypeptide that issubstantially free of cellular material includes preparations havingless than about 30%, 20%, 10%, 5% or 1% (by dry weight) of contaminatingprotein. When the polypeptide of the invention or biologically activeportion thereof is recombinantly produced, optimally culture mediumrepresents less than about 30% 20%, 10%, 5% or 1% (by dry weight) ofchemical precursors or non-protein-of-interest chemicals.

Fragments of the disclosed polynucleotides and polypeptides encodedthereby are also encompassed by the present invention. Fragments of apolynucleotide may encode polypeptide fragments that retain thebiological activity of the native polypeptide and hence are involved inthe metabolic conversion of nicotine to nornicotine in a plant.Alternatively, fragments of a polynucleotide that are useful ashybridization probes or PCR primers using methods described belowgenerally do not encode fragment polypeptides retaining biologicalactivity. Furthermore, fragments of the disclosed polynucleotidesinclude those that can be assembled within recombinant constructs foruse in gene silencing with any method known in the art, including, butnot limited to, sense suppression/cosuppression, antisense suppression,double-stranded RNA (dsRNA) interference, hairpin RNA interference andintron-containing hairpin RNA interference, amplicon-mediatedinterference, ribozymes and small interfering RNA or micro RNA, asdescribed in the art and herein below. Thus, fragments of apolynucleotide may range from at least about 20 nucleotides, about 50nucleotides, about 70 nucleotides, about 100 nucleotides about 150nucleotides, about 200 nucleotides, 250 nucleotides, 300 nucleotides andup to the full-length polynucleotide encoding the polypeptides of theinvention, depending upon the desired outcome. For example, thefragments of a polynucleotide can be between 100 and about 350nucleotides, between 100 and about 325 nucleotides, between 100 andabout 300 nucleotides, between about 125 and about 300 nucleotides,between about 125 and about 275 nucleotides in length, between about 200to about 320 contiguous nucleotides, between about 200 and about 420contiguous nucleotides in length between about 250 and about 450contiguous nucleotides in length. Alternatively, the fragment can bebetween about 300 and about 450 contiguous nucleotides in length.

A fragment of a nicotine demethylase polynucleotide of the presentinvention that encodes a biologically active portion of a cytochromeP450 polypeptide of the present invention will encode at least 15, 25,30, 50, 75, 100, 125, 150, 175, 200, 250, 300, 350, 400, 450 or 500contiguous amino acids, or up to the total number of amino acids presentin a full-length nicotine demethylase polypeptide of the invention(e.g., 517 amino acids for SEQ ID NOS:2 and 5), or will encode at least15, 25, 30, 50, 75, 100, 125, 150 or up to the total number of aminoacids present in a partial-length cytochrome P450 polypeptide of theinvention (e.g., 422 for SEQ ID NO:12). Preferably, the fragmentcomprises up to amino acid residue 330 of the encoded polypeptide. Abiologically active portion of a nicotine demethylase polypeptide can beprepared by isolating a portion of one of the cytochrome P450polynucleotides of the present invention, expressing the encoded portionof the cytochrome P450 polypeptide (e.g., by recombinant expression invitro), and assessing the activity of the encoded portion of thecytochrome P450 polypeptide, i.e., the ability to promote conversion ofnicotine to nornicotine, using assays known in the art and thoseprovided herein below.

Polynucleotides that are fragments of a cytochrome P450 nucleotidesequence of the present invention comprise at least 16, 20, 50, 75, 100,150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 800, 900,950, 1000, 1050, 1100, 1150, 1200, 1250, 1300, 1350, 1400, 1450, 1500,1550, 1600, 1650 or 1700 contiguous nucleic acids, or up to the numberof nucleotides present in a full-length cytochrome P450 polynucleotideas disclosed herein (e.g., 1554 for SEQ ID NO:1; or 2608 for SEQ IDNO:4). Polynucleotides that are fragments of a cytochrome P450nucleotide sequence of the present invention comprise fragments fromabout 20 to about 1700 contiguous nucleic acids, from about 50 to about1600 contiguous nucleic acids, from about 75 to about 1500 contiguousnucleic acids, from about 100 to about 1400 nucleic acids, from about150 to about 1300 contiguous nucleic acids, from about 150 to about 1200contiguous nucleic acids, from about 175 to about 1100 contiguousnucleic acids, from about 200 to about 1000 contiguous nucleic acids,from about 225 to about 900 contiguous nucleic acids, from about 500 toabout 1600 contiguous nucleic acids, from about 775 to about 1700contiguous nucleic acids, from about 1000 to about 1700 contiguousnucleic acids, or from about 300 to about 800 contiguous nucleic acidsfrom a cytochrome P450 polynucleotide as disclosed herein. For example,polynucleotide fragment can comprise a polynucleotide sequencecontaining the nucleic acid sequence from the polynucleotide at aboutposition 700 to about position 1250 of a cytochrome P450 codingsequence, at about position 700 to about position 1250 of a cytochromeP450 genomic sequence, at about position 10 to about position 900 of acytochrome P450 intron sequence, or at about position 100 to aboutposition 800 of a cytochrome P450 intron sequence.

Variants of the disclosed polynucleotides and polypeptides encodedthereby are also encompassed by the present invention. Naturallyoccurring variants include those variants that share substantialsequence identity to the disclosed cytochrome P450 polynucleotides andpolypeptides disclosed herein. Naturally occurring variants can sharesubstantial functional identity to the disclosed cytochrome P450polynucleotides disclosed herein. The compositions and methods of theinvention can be used to target expression or function of any naturallyoccurring cytochrome P450 that shares substantial sequence identity tothe disclosed cytochrome P450 polypeptides. Such cytochrome P450 canpossess the relevant cytochrome P450 activity, i.e., involvement in themetabolic conversion of nicotine to nornicotine in plants, or not. Suchvariants may result from, e.g., genetic polymorphism or from humanmanipulation as occurs with breeding and selection. Biologically activevariants of a cytochrome P450 protein of the invention, such as variantsof the polypeptide set forth in SEQ ID NO:2 and 5-24, will have at leastabout 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%,93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to the aminoacid sequence for the wild-type protein as determined by sequencealignment programs and parameters described elsewhere herein, and can becharacterized by a functional involvement in the metabolic conversion ofnicotine to nornicotine in plants or lack thereof. A biologically activevariant of a polypeptide of the invention may differ by as few as 1-15amino acid residues, as few as 10, as few as 9, as few as 8, as few as7, as few as 6, as few as 5, as few as 4, as few as 3, as few as 2, oras few as 1 amino acid residue from the wild-type polypeptide. Abiologically inactive variant of a protein of the invention may differfrom that polypeptide by as few as 1-15 amino acid residues, as few as10, as few as 9, as few as 8, as few as 7, as few as 6, as few as 5, asfew as 4, as few as 3, as few as 2, or as few as 1 amino acid residue.

Variants of the polynucleotides of the present invention include thosenaturally occurring polynucleotides that encode a nicotine demethylasepolypeptide that is involved in the metabolic conversion of nicotine tonornicotine in plants. Such polynucleotide variants can comprise adeletion and/or addition of one or more nucleotides at one or more siteswithin the native polynucleotide disclosed herein and/or a substitutionof one or more nucleotides at one or more sites in the nativepolynucleotide. Because of the degeneracy of the genetic code,conservative variants for polynucleotides include those sequences thatencode the amino acid sequence of one of the cytochrome P450polypeptides of the invention. Naturally occurring variants such asthese can be identified with the use of well-known molecular biologytechniques, as, e.g., with polymerase chain reaction (PCR) andhybridization techniques as are known in the art and disclosed herein.Variant polynucleotides also include synthetically derivedpolynucleotides, such as those generated, e.g., by using site-directedmutagenesis but which still share substantial sequence identity to thenaturally occurring sequences disclosed herein, and thus can be used inthe methods of the invention to inhibit the expression or function of anicotine demethylase that is involved in the metabolic conversion ofnicotine to nornicotine, including the nicotine demethylase polypeptidesset forth in SEQ ID NOS:2, 5, 7-11, 13, 16-21 and 23-24. Generally,variants of a particular polynucleotide of the invention, e.g., thepolynucleotide sequence of SEQ ID NO:3 or the polynucleotide sequenceencoding the amino acid sequence set forth in SEQ ID NO:2 and 5-24, willhave at least about 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%,90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequenceidentity to that particular polynucleotide as determined by sequencealignment programs and parameters described elsewhere herein.

Variants of a particular polynucleotide of the present invention (alsoreferred to as the reference polynucleotide) can also be evaluated bycomparison of the percent sequence identity between the polypeptideencoded by the reference polynucleotide and the polypeptide encoded by avariant polynucleotide. Percent sequence identity between any twopolypeptides can be calculated using sequence alignment programs andparameters described elsewhere herein. Where any given pair ofpolynucleotides of the invention is evaluated by comparison of thepercent sequence identity shared by the two polypeptides they encode,the percent sequence identity between the two encoded polypeptides is atleast about 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%,92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity. Avariant polypeptide of the present invention can include a polypeptidehaving a serine at position 458, an asparagine at position 364 of thecytochrome P450 polypeptide, a stop codon at position 329 of thecytochrome P450 or any combination thereof, where the numberingcorresponds to SEQ ID NO:13.

Furthermore, the polynucleotides of the invention can be used to isolatecorresponding cytochrome P450 sequences from other organisms,particularly other plants, more particularly other members of theNicotiana genus. PCR, hybridization and other like methods can be usedto identify such sequences based on their sequence homology to thesequences set forth herein. Sequences isolated based on their sequenceidentity to the nucleotide sequences set forth herein or to variants andfragments thereof are encompassed by the present invention. Suchsequences include sequences that are orthologs of the disclosedsequences.

As used herein, “orthologs” means genes derived from a common ancestralgene that are found in different species as a result of speciation.Genes found in different species are considered orthologs when theirnucleotide sequences and/or their encoded protein sequences share atleast 60%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,98%, 99% or greater sequence identity. Functions of orthologs are oftenhighly conserved among species. Thus, isolated polynucleotides thatencode for a nicotine demethylase polypeptide that is involved in thenicotine-to-nornicotine metabolic conversion and which hybridize understringent conditions to the cytochrome P450 sequences disclosed herein,or to variants or fragments thereof, are encompassed by the presentinvention. Such sequences can be used in the methods of the presentinvention to inhibit expression of nicotine demethylase polypeptidesthat are involved in the metabolic conversion of nicotine to nornicotinein plants.

Using PCR, oligonucleotide primers can be designed for use in PCRreactions to amplify corresponding DNA sequences from cDNA or genomicDNA extracted from any plant of interest. Methods for designing PCRprimers and PCR cloning are generally known in the art and are disclosedin Sambrook et al. (1989) Molecular cloning: a laboratory manual (2d ed,Cold Spring Harbor Laboratory Press, Plainview, N.Y.). Innis et al.,eds. (1990) PCR protocols: a guide to methods and applications (AcademicPress, New York); Innis & Gelfand, eds. (1995) PCR strategies (AcademicPress, New York); and Innis & Gelfand, eds. (1999) PCR methods manual(Academic Press, New York). Known methods of PCR include, but are notlimited to, methods using paired primers, nested primers, singlespecific primers, degenerate primers, gene-specific primers,vector-specific primers, partially mismatched primers and the like.

Hybridization techniques involve the use of all or part of a knownpolynucleotide as a probe that selectively hybridizes to othercorresponding polynucleotides present in a population of cloned genomicDNA fragments or cDNA fragments (i.e., genomic or cDNA libraries) from achosen organism.

Hybridization may be carried out under stringent conditions. As usedherein, “stringent conditions” or “stringent hybridization conditions”means conditions under which a probe will hybridize to its targetsequence to a detectably greater degree than to other sequences (e.g.,at least two-fold over background). Stringent conditions aresequence-dependent and will be different in different circumstances. Bycontrolling the stringency of the hybridization and/or washingconditions, target sequences that are 100% complementary to the probecan be identified (homologous probing). Alternatively, stringencyconditions can be adjusted to allow some mismatching in sequences sothat lower degrees of similarity are detected (heterologous probing).Generally, a probe is less than about 1000 nucleic acids in length,optimally less than 500 nucleic acids in length.

Typically, stringent conditions will be those in which the saltconcentration is less than about 1.5 M Na ion, typically about 0.01 to1.0 M Na ion concentration (or other salts) at pH 7.0 to 8.3 and thetemperature is at least about 30° C. Stringent conditions may also beachieved with the addition of destabilizing agents such as formamide.Exemplary low stringency conditions include hybridization with a buffersolution of 30 to 35% formamide, 1 M NaC1, 1% SDS (sodium dodecylsulphate) at 37° C., and a wash in 1×0 to 2× SSC (20× SSC =3.0 MNaCl/0.3 M trisodium citrate) at 50 to 55° C. Exemplary moderatestringency conditions include hybridization in 40 to 45% formamide, 1.0M NaCl 1% SDS at 37° C., and a wash in 0.5× to 1× SSC at 55 to 60° C.Exemplary high stringency conditions include hybridization in 50%formamide, 1 M NaCl, 1% SDS at 37° C. and a wash in 0.1× SSC at 60 to65° C. Optionally, wash buffers may comprise about 0.1% to about 1% SDS.Duration of hybridization is generally less than about 24 hours, usuallyabout 4 to about 12 hours. The duration of the wash time will be atleast a length of time sufficient to reach equilibrium.

Preferably, stringency conditions include hybridization in a solutioncontaining 5× SSC, 0.5% SDS, 5× Denhardt's, 0.45 ug/ul poly A RNA, 0.45ug/ul calf thymus DNA and 50% formamide at 42° C., and at least onepost-hybridization wash in a solution comprising from about 0.01× SSC toabout 1× SSC. The duration of hybridization is from about 14 to about 16hours.

Specificity is typically the function of post-hybridization washes, thecritical factors being the ionic strength and temperature of the finalwash solution. For DNA-DNA hybrids, the T_(m) can be approximated fromthe equation of Meinkoth & Wahl (1984) Anal. Biochem. 138:267-284:T_(m)=81.5° C.+16.6 (log M)+0.41 (% GC)−0.61 (% form)−500/L; where M isthe molarity of monovalent cations, % GC is the percentage of guanosineand cytosine nucleotides in the DNA, % form is the percentage offormamide in the hybridization solution, and L is the length of thehybrid in base pairs. The T_(m) is the temperature (under defined ionicstrength and pH) at which 50% of a complementary target sequencehybridizes to a perfectly matched probe. T_(m) is reduced by about 1° C.for each 1% of mismatching; thus, T_(m), hybridization, and/or washconditions can be adjusted to hybridize to sequences of the desiredidentity. For example, if sequences with ≥90% identity are sought, theT_(m) can be decreased 10° C. Generally, stringent conditions areselected to be about 5° C. lower than the thermal melting point (T_(m))for the specific sequence and its complement at a defined ionic strengthand pH. However; severely stringent conditions can utilize ahybridization and/or wash at 1, 2, 3 or 4° C. lower than the thermalmelting point (T_(m)); moderately stringent conditions can utilize ahybridization and/or wash at 6, 7, 8, 9 or 10° C. lower than the thermalmelting point (T_(m)); low stringency conditions can utilize ahybridization and/or wash at 11, 12, 13, 14, 15 or 20° C. lower than thethermal melting point (T_(m)). Using the equation, hybridization andwash compositions, and desired T_(m), those of ordinary skill willunderstand that variations in the stringency of hybridization and/orwash solutions are inherently described. If the desired degree ofmismatching results in a T_(m), of less than 45° C. (aqueous solution)or 32° C. (formamide solution), it is optimal to increase the SSCconcentration so that a higher temperature can be used. An extensiveguide to the hybridization of nucleic acids is found in Tijssen (1993)Laboratory techniques in biochemistry and molecularbiology-hybridization with nucleic acid probes, Part I, Chapter 2(Elsevier, New York); and Ausubel et al., eds. (1995) Current protocolsin molecular biology, Chapter 2 (Greene Publishing andWiley-Interscience, New York). See also, Sambrook et al. (1989)Molecular cloning: a laboratory manual (2d ed., Cold Spring HarborLaboratory Press, Plainview, N.Y.).

Hybridization probes may be genomic DNA fragments, cDNA fragments, RNAfragments or other oligonucleotides, and may be labeled with adetectable group such as ³²P, or any other delectable marker. Forexample, probes for hybridization can be made by labeling syntheticoligonucleotides based on the cytochrome P450 polynucleotides sequencesof the present invention. Methods for preparation of probes forhybridization and for construction of cDNA and genomic libraries aregenerally known in the art and are disclosed in Sambrook et al., supra.

For example, the cytochrome P430 polynucleotides disclosed herein, orone or more portions thereof, may be used as probes capable ofspecifically hybridizing to corresponding cytochrome P450polynucleotides and messenger RNAs. To achieve specific hybridizationunder a variety of conditions, such probes include sequences that areunique among cytochrome P450 polynucleotide sequences or unique to oneof the cytochrome P450 polynucleotide sequences, including upstreamregions 5′ to the coding sequence and downstream regions 3′ to thecoding sequence and an intron region, and are optimally at least about10 contiguous nucleotides in length, more optimally at least about 20contiguous nucleic acids in length, more optimally at least about 50contiguous nucleic acids in length, more optimally at least about 75contiguous nucleic acids in length, and more optimally at least about100 contiguous nucleic acids in length. Such probes may be used toamplify corresponding cytochrome P450 polynucleotides. This techniquemay be used to isolate additional coding sequences or mutations from adesired plant or as a diagnostic assay to determine the presence ofcoding sequences in a plant. Hybridization techniques includehybridization screening of plated DNA libraries (either plaques orcolonies; see, e.g., Sambrook et al., supra.

As used herein, with respect to the sequence relationships between twoor more polynucleotides or polypeptides, the term “reference sequence”means a defined sequence used as a basis for sequence comparison. Areference sequence may be a subset or the entirety of a specifiedsequence; for example, as a segment of a full-length cDNA or genesequence, or the complete cDNA or gene sequence.

As used herein, “comparison window” means a contiguous and specifiedsegment of a polynucleotide sequence, where the polynucleotide sequencein the comparison window may comprise additions or deletions (i.e.,gaps) compared to the reference sequence (which does not compriseadditions or deletions) for optimal alignment of the twopolynucleotides. Generally, a comparison window is at least 20contiguous nucleic acids in length, and optionally can be 30, 40, 50 or100 contiguous nucleic acids or longer. Those of skill in the artunderstand that to avoid a high similarity to a reference sequence dueto inclusion of gaps in the polynucleotide sequence a gap penalty istypically introduced and is subtracted from the number of matches.

Methods of alignment of sequences for comparison are well known in theart. Thus, the determination of percent sequence identity between anytwo sequences can be accomplished using a mathematical algorithm.Non-limiting examples of such mathematical algorithms are the algorithmof Myers & Miller (1988) CABIOS 4:11-17; the local alignment algorithmof Smith et al. (1981) Adv. Appl. Math. 2:482; the global alignmentalgorithm of Needleman & Wunsch (1970) J. Mol. Biol. 48:443-453; thesearch-for-local alignment method of Pearson & Lipman (1988) Proc. Natl.Acad. Sci. 85:2444-2448; the algorithm of Karlin & Altschul (1990) Proc.Natl. Acad. Sci. USA 872264, modified as in Karlin & Altschul (1993)Proc. Natl. Acad. Sci. USA 90:5873-5877.

The BLAST programs of Altschul et al. (1990) J. Mol. Biol. 215:403 arebased on the algorithm of Karlin & Altschul (1990) supra. BLASTnucleotide searches can be performed with the BLASTN program, score=100,wordlength=12, to obtain nucleotide sequences homologous to a nucleotidesequence encoding a protein of the invention. BLAST protein searches canbe performed with the BLASTX program, score =50. wordlength=3, to obtainamino acid sequences homologous to a protein or polypeptide of theinvention. To obtain gapped alignments for comparison purposes, GappedBLAST (in BLAST 2.0) can be utilized as described in Altschul et al.(1997) Nucleic Acids Res. 25:3389. Alternatively, PSI-BLAST (in BLAST2.0) can be used to perform an iterated search that detects distantrelationships between molecules. See Altschul et al. (1997) supra. Whenutilizing BLAST, Gapped BLAST, PSI-BLAST, the default parameters of therespective programs (e.g., BLASTN for nucleotide sequences, BLASTX forproteins) can be used (See www.ncbi.nlna.nih.gov). Alignment may also beperformed manually by inspection.

The sequence identity/similarity values provided herein were calculatedusing the BLASTX (Altschul et al. (1997) supra), Clustal W (Higgins etal. (1994) Nucleic Acids Res. 22:4673-4680), and GAP (University ofWisconsin Genetic Computing Group software package) algorithms usingdefault parameters. The present invention also encompasses the use ofany equivalent program thereof for the analysis and comparison ofnucleic acid and protein sequences. By “equivalent program” is intendedany sequence comparison program that, for any two sequences in question,generates an alignment having identical nucleotide or amino acid residuematches and an identical percent sequence identity when compared to thecorresponding alignment generated by BLASTX. Clustal W, or GAP.

For purposes of the foregoing discussion of variant nucleotide andpolypeptide sequences encompassed by the present invention, “sequenceidentity” or “identity” in the context of two polynucleotides orpolypeptide sequences makes reference to the residues in the twosequences that are the same when aligned for maximum correspondence overa specified comparison window. When percentage of sequence identity isused in reference to proteins it is recognized that residue positionswhich are not identical often differ by conservative amino acidsubstitutions, where amino acid residues are substituted for other aminoacid residues with similar chemical properties (e.g., charge orhydrophobicity) and therefore do not change the functional properties ofthe molecule. When sequences differ in conservative substitutions, thepercent sequence identity may be adjusted upwards to correct for theconservative nature of the substitution. Sequences that differ by suchconservative substitutions are said to have “sequence similarity” or“similarity.” Means for malting this adjustment are well known to thoseof skill in the art. Typically this involves scoring a conservativesubstitution as a partial rather than a full mismatch, therebyincreasing the percentage sequence identity. Thus, for example, where anidentical amino acid is given a score of 1 and a non-conservativesubstitution is given a score of zero, a conservative substitution isgiven a score between zero and 1. The scoring of conservativesubstitutions is calculated, e.g., as implemented in the program PC/GENE(Intelligenetics; Mountain View, Calif.).

As used herein, “percentage of sequence identity” means the valuedetermined by comparing two optimally aligned sequences over acomparison window, where the portion of the polynucleotide sequence inthe comparison window may comprise additions or deletions (i.e., gaps)as compared to the reference sequence (which does not comprise additionsor deletions) for optimal alignment of the two sequences. The percentageis calculated by determining the number of positions at which theidentical nucleic acid base or amino acid residue occurs in bothsequences to yield the number of matched positions, dividing the numberof matched positions by the total number of positions in the window ofcomparison, and multiplying the result by 100 to yield the percentage ofsequence identity.

Thus, cytochrome P450 polynucleotide and polypeptide sequences can beidentified using the sequences provided herein. Such methods includeobtaining a polynucleotide or polypeptide sequence at least 80%, 85%,90%, 95%, 98% or 99% sequence identity with the polynucleotide sequenceof SEQ ID NO:1, 3 or 4 or a complement or fragment thereof, or apolypeptide sequence of SEQ ID NO:2 or 5-24. A preferred embodimentincludes a polypeptide corresponding to SEQ ID NO:13 that has a serineat position 458 or an asparagine at position 364 of the cytochrome P450polypeptide, or a stop codon at position 329 of the cytochrome P450, ora combination thereof, where the numbering corresponds to SEQ ID NO:13.

Expression Cassettes for Use in the Methods of Invention

Compositions of the present invention further include expressioncassettes comprising inhibitory sequences capable of inhibitingexpression or function of a nicotine demethylase polypeptide involved inthe conversion of nicotine to nornicotine in a Nicotiana plant or plantpart thereof, where the inhibitory sequences are operably linked to apromoter that is functional in a plant cell. In this manner, expressioncassettes comprising all or part of the sequence set forth in SEQ IDNO:1, 3 or 4 or encoding SEQ ID NO:2 or 5-24, a complement or fragmentthereof, or sequences sharing substantial sequence identity to suchsequences, or a complement or fragment thereof, operably linked to apromoter that is functional in a plant cell are constructed for use inthe gene-silencing methods of the present invention described hereinbelow. Such sequences are referred to herein as “inhibitory sequences”or “inhibitory polynucleotide sequences,” as they are capable of beingexpressed as an RNA molecule that inhibits expression (i.e.,transcription and/or translation) of the target cytochrome P450polypeptide, for example, the polypeptide set forth in SEQ ID NO:2 or5-24 and variants thereof, where the variant polypeptides havesubstantial sequence identity to these disclosed cytochrome P450polypeptides. Such variants may or may not be involved in the metabolicconversion of nicotine to nornicotine in a plant. Such sequences alsoinclude fragment sequences of the target cytochrome P450 polynucleotideor polypeptide. For example, a fragment sequence can include any portionof the cytochrome P450 sequence, including coding and non-codingsequence (e.g., 5′ UTR, intron, and 3′ UTR sequences), and can includefragments between 100 and about 350 nucleic acids, between about 125 andabout 300 nucleic acids, or between about 125 and about 275 nucleicacids. Preferably, a fragment of nicotine demethylase can be betweenabout 20 and about 420, about 30 and about 420, between about 40 andabout 320, between about 50 and about 200, between about 50 and about400, between about 50 and about 420, between about 60 and about 320,about 70 and about 220, between about 100 and about 200, between about100 and about 320, between about 150 and about 200, between about 150and about 220, between about 150 and about 400, between about 200 andabout 300, or between about 300 and about 400 contiguous nucleic acids.Alternatively, a fragment of a cytochrome P450 can be about 100, about150, about 200, about 220, about 250, about 300, about 320, or about 350contiguous nucleic acids in length. Alternatively yet, a cytochrome P450fragment can be reduced in length by about 20, about 40, about 60, about80, about 100, about 120, about 140, about 160, about 180, about 200,about 220, about 240, about 260, about 280, about 290, about 300, about320, about 340, about 360, about 380, about 400 contiguous nucleic acidscompared to the full-length. For all of these cytochrome P450 fragments,the truncation or deletion can start at the 5′ end, start at the 3′ end,or be internal to a cytochrome P450 or a cytochrome P450 intron. For acytochrome P450 intron fragment, the entire sequence of a cytochromeP450 intron can be SEQ ID NO:3.

Furthermore, a fragment of a cytochrome P450 polynucleotide orpolypeptide can contain contiguous nucleotides from about 1, 2, 5, 10,20, 30, 40, 50, 60, 70, 80, or 90% of the entire gene. Alternativelystated, a fragment of a cytochrome P450 polynucleotide or polypeptidecan be between about 5% - about 80%, between about 10%—about 70%,between about 10%—about 60%, between about 10%—about 50%, between about25%—about 60%, between about 25%—about 50%, between about 40%—about 60%,between about 40%—about 80%, between about 50%—about 90% of the lengthof an entire cytochrome P450.

Expression cassettes of the present invention include those thatencompass additional domains that modulate the level of expression, thedevelopmental timing of expression, or tissue type that expressionoccurs in (e.g., AU Patent No. AU-A-77751/94 and U.S. Pat. Nos.5,466,785 and 5,635,618). Promoters can be selected based on the desiredoutcome. The nucleic acids of the present invention can be combined withinducible, constitutive, pathogen- or wound-induced, environmentally- ordevelopmentally-regulated, cell- or tissue-preferred promoter, or otherpromoters for expression in plants.

Chemical-inducible promoters can be used to inhibit the expression of acytochrome P450 that is involved in the metabolic conversion of nicotineto nornicotine in a plant through the application of an exogenouschemical regulator. Chemical-inducible promoters are known in the artand include, but are not limited to, the tobacco PR-la promoter, whichis activated by salicylic acid. Other chemical-inducible promoters ofinterest include steroid-responsive promoters (see, e.g., theglucocorticoid-inducible promoter (Schena et al. (1991) Proc. Natl.Acad. Sci. USA 88:10421-10425; and McNellis et al. (1998) Plant J.14(2):247-257) and tetracycline-inducible promoters (see, e.g., Gatz etal. (1991) Mol. Gen. Genet. 227:229-237; and U.S. Pat. Nos. 5,814,618and 5,789,156), each of which is incorporated herein by reference as ifset forth in its entirety.

Constitutive promoters include, e.g., the core promoter of the Rsyn7promoter and outer constitutive promoters disclosed in U.S. Pat. No.6,072,050; the core CaMV 35S promoter (Odell et al. (1985) Nature313:810-812); ubiquitin (Christensen et al. (1989) Plant Mol. Biol.12:619-632; and Christensen et al. (1992) Plant Mol. Biol. 18:675-689);pEMU (Last et al. (1991) Theor. Appl. Genet. 81:581-588); MAS (Velten etal. (1984) EMBO J. 3:2723-2730); ALS promoter (U.S. Pat. No. 5,659,026),and the like. Other constitutive promoters include, e.g., thosedisclosed in U.S. Pat. Nos. 5,608,149; 5,608,144; 5,604,121; 5,569,597;5,466,785; 5,399,680; 5,268,463; 5,608,142 and 6,177,611.

Tissue-preferred promoters can be utilized to target expression of aninhibitory polynucleotide sequence of the present invention within aparticular plant tissue. Tissue-preferred promoters include thosedisclosed in Yamamoto et al. (1997) Plant J. 12(2):255-265; Kawamata etal. (1997) Plant Cell Physiol. 38(7):792-803; Hansen et al. (1997) Mol.Gen Genet. 254(3):337-343; Russell et al. (1997) Transgenic Res.6(2):157-168; Rinehart et al. (1996) Plant Physiol. 112(3):1331-1341;Van Camp et al. (1996) Plant Physiol. 112(2):525-535; Canevascini et al.(1996) Plant Physiol. 112(2):513-524; Yamamoto et al. (1994) Plant CellPhysiol. 35(5):773-778; Lam (1994) Results Probl. Cell Differ.20:181-196; Orozco et al. (1993) Plant Mol Biol. 23(6):1129-1138;Matsuoka et al. (1993) Proc Natl. Acad. Sci. USA 90(20):9586-9590; andGuevara-Garcia et al. (1993) Plant J. 4(3):495-505.

Of particular interest herein are leaf preferred promoters that providefor expression predominately in leaf tissues. See, e.g., Yamamoto et al.(1997) Plant 12(2):255-265; Kwon et al. (1994) Plant Physiol.105:357-67; Yamamoto et al. (1994) Plant Cell Physiol. 35(5):773-778;Gotor et al. (1993) Plant J. 3:509-18; Orozco et al. (1993) Plant Mol.Biol. 23(6):1129-1138; Baszczynski et al. (1988) Nucl. Acid Res.16:4732; Mitra et al. (1994) Plant Molecular Biology 26:35-93; Kayaya etal. (1995) Molecular and General Genetics 248:668-674; and Matsuoka etal. (1993) Proc. Natl. Acad. Sci. USA 90(20):9586-9590.Senecence-regulated et al. (1998) Plant Physiol. 116:329-335); SAG 13(Gan and Amasino (1997) Plant Physiol. 113:313-319; SAG 15 (Gan (1995)“Molecular Characterization and Genetic Manipulation of PlantSenescence,” Ph.D. Thesis, University of Wisconsin, Madison); SENT (Ohet al. (1996) Plant Mol. Biol. 30:739-754; promoter of asenescence-specific gene for expression of IPT (Gan and Amasino 91995)Science 270:1986-1988); and the like (see, e.g., Or et al. (1999) PlantCell 11:1073-1080; and McCabe et al. (2001) Plant Physiol. 127:505-516).

Expression cassettes of the present invention can include 5′ leadersequences that can act to enhance translation. Translation leaders areknown in the art and include, but are not limited to, picornavirusleaders, e.g., EMCV leader (Encephalomyocarditis 5′ noncoding region;Elroy-Stein et al. (1989) Proc. Natl. Acid. Sci. USA 86:6126-6130);potyvirus leaders, e.g., TEV leader (Tobacco Etch Virus; Gallie et al.(1995) Gene 165(2):233-238), MDMV leader (Maize Dwarf Mosaic Virus;Virology 154:9-20), and human immunoglobulin heavy-chain binding protein(BiP; Macejak et al. (1991) Nature 353:90-94); untranslated leader fromthe coat protein mRNA of alfalfa mosaic virus (AMV RNA 4; Jobling et al.(1987) Nature 325:622-625); tobacco mosaic virus leader (TMV; Gallie etal. (1989) in Molecular biology of RNA, ed. Cech (Liss, New York), pp.237-256); and maize chlorotic mottle virus leader (MCMV; Lommel et al.(1991) Virology 81:382-385). See also, Della-Cioppa et al. (1987) PlantPhysiol. 84:965-968. Other methods known to enhance translation also canbe utilized.

Methods for Inhibiting Expression or Function of a Nicotine Demethylase

Methods of reducing the concentration, content and/or activity of acytochrome P450 polypeptide of the present invention in a Nicotianaplant or plant part, particularly the leaf tissue, are provided. Manymethods may be used, alone or in combination, to reduce or eliminate theactivity of a cytochrome P450 polypeptide of the present invention, moreparticularly, the CYP82E5v2 nicotine demethylase. In addition,combinations of methods may be employed to reduce or eliminate theactivity of two or more different cytochrome P450 polypeptides, moreparticularly the CYP82E5v2 and CYP82E4v2 nicotine demethylases.Preferably, the CYP82E5v2 is a polypeptide with at least one amino acidmutation in SEQ ID NO:2 that negatively affects conversion in greenleaves and the CYP82E4v2 has the sequence set forth in SEQ ID NO:13 withat least one amino acid mutation that negatively affects conversion insenescent leaves.

In accordance with the present invention, the expression of a cytochromeP450 polypeptide of the present invention is inhibited if the proteinlevel of the cytochrome P450 polypeptide is statistically lower than theprotein level of the same cytochrome P450 polypeptide in a plant thathas not been genetically modified or mutagenized to inhibit theexpression of that cytochrome P450 polypeptide, and where these plantshave been cultured and harvested using the same protocols. In particularembodiments of the invention, the protein level of the cytochrome P450polypeptide in a modified plant according to the invention is less than95%, less than 90%, less than 80%, less than 70%, less than 60%, lessthan 50%, less than 40%, less than 30%, less than 20%, less than 10%, orless than 5% of the protein level of the same cytochrome P450polypeptide in a plant that is not a mutant or that has not beengenetically modified to inhibit the expression of that cytochrome P450polypeptide and which has been cultured and harvested using the sameprotocols. The expression level of the cytochrome P450 polypeptide maybe measured directly, for example, by assaying for the level of thecytochrome P450 transcript or cytochrome P450 polypeptide expressed inthe Nicotiana plant or plant part, or indirectly, e.g., by measuring theconversion of nicotine to nornicotine in the Nicotiana plant or plantpart. Methods for monitoring expression level of a polypeptide are knownin the art, and include, but are not limited to, Northern blot analysisand RNA differentiation assays. Methods for determining the activity ofa targeted cytochrome P450 polypeptide in converting nicotine tonornicotine are known in the art and described elsewhere herein below,and include, but are not limited to, alkaloid analysis using gaschromatography.

In some instances, the activity of one or more cytochrome P450polypeptides is reduced or eliminated by transforming a plant or plantpart with an expression cassette comprising a polynucleotide encoding apolypeptide that inhibits the activity of one or more cytochrome P450polypeptides of the present invention. A number of approaches have beenused to combine transgenes or mutations in one plant—including sexualcrossing, retransformation, co-transformation and the use of linkedtransgenes. A chimeric transgene with linked partial gene sequences canbe used to coordinately suppress numerous plant endogenous genes.Constructs modeled on viral polyproteins can be used to simultaneouslyintroduce multiple coding genes into plant cells. For a review, seeHalpin et al., Plant Mol. Biol. 47:295-310 (2001). A plant having amutation in CYP82E4v2 that inhibits the nicotine demethylase activity insenescent leaves can be crossed with a plant having a mutation inCYP83E5v2 that inhibits nicotine demethylase in green leaves to producea plant with conversion levels lower than about 0.2%, 0.3%, 0.4%, 0.5%,0.6% or 0.7%. Alternatively, a plant having one or more mutations inCYP82E4v2 at position 458, 364, 38, 201, 169, 459, 296, 427, 329, 376 or171 can be crossed with a plant having one or more mutations inCYP83E5v2 at position 235, 449, 174, 410, 224, 72 or 143 to produce aplant with conversion levels lower than 0.2%, 0.3%, 0.4%, 0.5%, 0.6% or0.7%. Alternatively still, a plant having one or more mutations inCYP82E4v2 at position 458, 364 or 329 can be crossed with a plant havingone or more mutations in CYP83E5v2 at position 235, 449, 174, 410, 224,72, or 143 to produce a plant with conversion levels lower than 0.2%,0.3%, 0.4%, 0.5%, 0.6%, or 0.7%. A particularly preferred conversionlevel of nicotine to nornicotine can be between 0.05%-0.4%, between0.1-0.6%, between 0.1%-0.3%, between 0.1%-0.5%, between 0.1%-0.4%,between 0.1%-0.7%, or between 0.1%-1.0%. Any mutation of apolynucleotide of the present invention that results in a truncation ofthe CYP83E4v2 or CYP83E5v2 polypeptide before a conserved heme-bindingmotif will inhibit the enzyme and can be used in a cross describedabove. The domains of cytochrome P450 proteins are known in the art.See, e.g., Xu et al. (2007) Physiologia Plantarum 129:307-319,incorporated herein by reference as if set forth in its entirety. Bycrossing plants having a nonfunctional or inhibited CYP82E4v2 gene, anonfunctional or inhibited CYP82E5v2 gene, or nonfunctional or inhibitedCYP82E4v2 and CYP82E5v2 genes, nornicotine levels can be reduced in atobacco plant.

The activity of a nicotine demethylase polypeptide in convertingnicotine to nornicotine in a Nicotiana plant or plant part is inhibitedaccording to the present invention if this conversion activity isstatistically lower than conversion activity of the same cytochrome P450polypeptide in a Nicotiana plant or plant part that has not beengenetically modified to inhibit the conversion activity of thatcytochrome P450 polypeptide and that has been cultured and harvestedusing the same protocols. In particular embodiments, activity of acytochrome P450 polypeptide in converting nicotine to nornicotine in amodified Nicotiana plant or plant part according to the invention isinhibited if the activity is less than 95%, less than 90%, less than 80%less than 70%, less than 60%, less than 50%, less than 40%, less than30%, less than 20% less than 10%, less than 5%, less than 2% or lessthan 1% of the conversion activity of the same cytochrome P450polypeptide in a Nicotiana plant that that has not been geneticallymodified to inhibit the expression of that nicotine demethylasepolypeptide and that has been cultured and harvested using the sameprotocols. The activity of a nicotine demethylase polypeptide inconverting nicotine to nornicotine in a Nicotiana plant or plant part iseliminated according to the invention when it is not detectable by theassay methods known in the art or described herein. Methods ofdetermining the activity of a cytochrome P450 polypeptide in convertingnicotine to nornicotine in a Nicotiana using gas chromatography aredisclosed in the examples below.

In specific embodiments, a cytochrome P450 inhibitory polynucleotide ornicotine demethylase mutation described herein is introduced into aNicotiana plant or plant part. Subsequently, a Nicotiana plant or plantpart having the introduced inhibiting polynucleotide of the presentinvention is selected using methods known to those of skill in the artincluding, but not limited to, Southern blot analysis, DNA sequencing,PCR analysis or phenotypic analysis. A polynucleotide or polypeptidesequence of the present invention includes full-length polynucleotide orpolypeptide sequences, truncated polynucleotide or polypeptidesequences, fragments of polynucleotide or polypeptide sequences,variants of polynucleotide or polypeptide sequences, sense-orientednucleotide sequences, antisense-oriented nucleotide sequences, thecomplement of a sense- or antisense-oriented nucleotide sequence,inverted regions of nucleotide sequences, hairpins of nucleotidesequences, double-stranded nucleotide sequences, single-strandednucleotide sequences, combinations thereof, and the like. A plant orplant part altered or modified by the foregoing is grown underplant-forming conditions for a time sufficient to modulate theconcentration and/or activity of polypeptides of the present inventionin the plant. Plant forming conditions are well known in the art anddiscussed briefly elsewhere herein.

A transformed tobacco plant containing a nicotine demethylase inhibitorypolynucleotide sequence described herein has a reduced level ofconversion of nicotine to nornicotine. In particular embodiments,conversion of nicotine to nornicotine in a transformed tobacco plant orplant part according to the invention is less than 95%, less than 90%,less than 80% less than 70%, less than 60%, less than 50%, less than40%, less than 30%, less than 20%, less than 10%, less than 5%, lessthan 2% or less than 1% of the conversion in a tobacco plant that thathas not been genetically modified to inhibit the expression of thatnicotine demethylase polypeptide and with has been cultured andharvested using the same protocols. In some instances, the transformedtobacco plant is a converter tobacco plant. Alternatively, thetransformed tobacco plant is a nonconverter tobacco plant. Alternativelystill, the transformed tobacco plant has a conversion rate lower thanthe rate observed in commercial non-converter tobacco plants.

The level and/or activity of the polypeptide may be modulated byemploying a polynucleotide that is not capable of directing, in atransformed plant, the expression of a protein or an RNA. For example,the polynucleotides of the invention may be used to designpolynucleotide constructs that can be employed in methods for alteringor mutating a genomic nucleotide sequence in an organism. Suchpolynucleotide constructs include, but are not limited to, RNA:DNAvectors, RNA:DNA mutational vectors, RNA:DNA repair vectors,mixed-duplex oligonucleotides, self-complementary RNA:DNAoligonucleotides and recombinogenic oligonucleobases. Such nucleotideconstructs and methods of use are known in the art. See, U.S. Pat. Nos.5,565,350; 5,731,181; 5,756,325; 5,760,012; 5,795,972 and 5,871,984;each of which is incorporated herein by reference as if set forth in itsentirety. See also, International Patent Application Publication Nos. WO98/149350, WO 99/107865 and WO 99/125921; and Beetham et al. (1999)Proc. Natl. Acad. Sci. USA 96:8774-8778; each of which is incorporatedherein by reference as if set forth in its entirety.

The methods of the present invention do not depend on the incorporationof the entire nicotine demethylase inhibitory polynucleotide into thegenome, only that the Nicotiana plant or plant part thereof is alteredas a result of the introduction of this inhibitory polynucleotide into acell. As such, the genome may be altered following the introduction ofthe nicotine demethylase inhibitory polynucleotide into a cell. Forexample, the inhibitory polynucleotide, or any part thereof, mayincorporate into the genome of the plant. Alterations to the genomeinclude, but are not limited to, additions, deletions and substitutionsof nucleotides into the genome. While the methods of the presentinvention do not depend on additions, deletions and substitutions of anyparticular number of nucleotides, it is recognized that such additions,deletions and/or substitutions comprise at least one nucleotide.

Further, one can reduce the level and/or activity of a nicotinedemethylase sequence by eliciting the effects of the sequence onlyduring certain developmental stages and to switch the effect off inother stages where expression is no longer desirable. Control ofnicotine demethylase expression can be obtained via the use of inducibleor tissue-preferred promoters. Alternatively, the gene could be invertedor deleted using site-specific recombinases, transposons orrecombination systems, which would also turn on or off expression of thecytochrome P450 sequence.

According to the present invention, changes in levels, ratios, activityor distribution of cytochrome P450 polypeptides of the presentinvention, or changes in Nicotiana plant or plant part phenotype,particularly reduced accumulation of nornicotine and its carcinogenicmetabolite, NNN, can be measured by comparing a subject plant or plantpart to a control plant or plant part, where the subject plant or plantpart and the control plant or plant part have been cultured and/orharvested using the same protocols. As used herein, a subject plant orplant part is one in which genetic alteration, such as transformation,has been affected as to the nicotine demethylase polypeptide ofinterest, or is a Nicotiana plant or plant part that is descended from aNicotiana plant or plant part so altered and which comprises thealteration. A control plant or plant part provides a reference point formeasuring changes in phenotype of the subject plant or plant part. Themeasurement of changes in phenotype can be measured at any time in aplant or plant part, including during plant development, senescence, orafter curing. In other embodiments, the measurement of changes inphenotype can be measured in plants grown under any conditions,including from plants grown in growth chamber, greenhouse, or in afield. In one embodiment, changes in phenotype can be measured bydetermining the nicotine to nornicotine conversion rate. In a preferredembodiment, conversion can be measured by dividing the percentage ofnornicotine (as a percentage of the total tissue weight) by the sum ofthe percentage nicotine and nornicotine (as percentages of the totaltissue weight) and multiplying by 100.

According to the present invention, a control plant or plant part maycomprise a wild-type Nicotiana plant or plant part, i.e., of the samegenotype as the starting material for the genetic alteration thatresulted in the subject plant or plant part. A control plant or plantpart may also comprise a Nicotiana plant or plant part of the samegenotype as the starting material but that has been transformed with anull construct (i.e., with a construct that has no known effect on thetrait of interest, such as a construct comprising a selectable markergene). Alternatively, a control plant or plan part may comprise aNicotiana plant or plant part that is a non-transformed segregant amongprogeny of a subject plant or plant part, or a Nicotiana plant or plantpart genetically identical to the subject plant or plant part but thatis not exposed to conditions or stimuli that would induce suppression ofthe nicotine demethylase gene of interest. Finally, a control plant orplant part may comprise the subject plant or plant part itself underconditions in which the nicotine demethylase inhibitory sequence is notexpressed. In all such cases, the subject plant or plant part and thecontrol plant or plant part are cultured and harvested using the sameprotocols.

As described elsewhere herein, methods are provided to reduce oreliminate the activity and/or concentration of a nicotine demethylasepolypeptide of the present invention by introducing into a Nicotianaplant or plant part a nicotine demethylase inhibitory polynucleotidesequence than is capable of inhibiting expression or function of anicotine demethylase polypeptide that is involved in the metabolicconversion of nicotine to nornicotine. In some instances, the inhibitorysequence can be introduced by transformation of the plant or plant partsuch as a plant cell with an expression cassette that expresses apolynucleotide that inhibits the expression of the nicotine demethylasepolypeptide. The polynucleotide may inhibit the expression of a nicotinedemethylase polypeptide directly, by preventing translation of thenicotine demethylase polypeptide messenger RNA, or indirectly, byencoding a polypeptide that inhibits the transcription or translation ofa nicotine demethylase polypeptide gene encoding a nicotine demethylasepolypeptide. Methods for inhibiting or eliminating the expression of agene product in a plant are well known in the art, and any such methodmay be used in the present invention to inhibit the expression ofnicotine demethylase polypeptides.

In other embodiments, the activity of a nicotine demethylase polypeptideof the present invention may be reduced or eliminated by disrupting thegene encoding the nicotine demethylase polypeptide. The inventionencompasses mutagenized plants that carry mutations in cytochrome P450genes, where the mutations reduce expression of the nicotine demethylasegene or inhibit the activity of an encoded nicotine demethylasepolypeptide of the present invention.

To obtain the desired plants, a Nicotiana plant or plant part can betransformed with an expression cassette that is capable of expressing apolynucleotide that inhibits the expression of a nicotine demethylasesequence. Such methods may include the use of sensesuppression/cosuppression, antisense suppression, double-stranded RNA(dsRNA) interference, hairpin RNA interference and intron-containinghairpin RNA interference, amplicon-mediated interference, ribozymes, andsmall interfering RNA or micro RNA.

For cosuppression, an expression cassette is designed to express an RNAmolecule corresponding to all or part of a messenger RNA encoding acytochrome P450 polypeptide of interest (for example, a cytochrome P450polypeptide comprising the sequence set forth in SEQ ID NO:2 or 5-24 ora sequence having substantial sequence identity to SEQ ID NO:2 or 5-24)in the “sense” orientation. Over-expression of the RNA molecule canresult in reduced expression of the native gene. Multiple plant linestransformed with the cosuppression expression cassette are then screenedto identify those that show the greatest inhibition of nicotinedemethylase polypeptide expression.

The polynucleotide used for cosuppression may correspond to all or partof the sequence encoding a cytochrome P450 polypeptide or the presentinvention, all or part of the 5′ and/or 3′ untranslated region of acytochrome P450 polypeptide transcript, or all or part of both thecoding sequence and the untranslated regions of a transcript encoding acytochrome P450 polypeptide. In some embodiments where thepolynucleotide comprises all or part of the coding region for acytochrome P450 polypeptide of the present invention, the expressioncassette can be designed to eliminate the start codon of thepolynucleotide so that no polypeptide product will be transcribed.

Cosuppression may be used to inhibit the expression of plant genes toproduce plants having undetectable levels for the polypeptides encodedby these genes or may also be used to inhibit the expression of multipleproteins in the same plant (e.g., Broin et al. (2002) Plant Cell14:1417-1432; and U.S. Pat. No. 5,942,657). Methods for usingcosuppression to inhibit the expression of endogenous genes in plantsare described in Flavell et al. (1994) Proc Natl. Acad. Sci. USA91:3490-3496; Jorgensen et al. (1996) Plant Mol. Biol. 31:957-973;Johansen and Carrington (2001) Plant Physiol. 126:930-938; Brain et al.(2002) Plant Cell 14:1417-1432; Stoutjesdijk et al. (2002) PlantPhysiol. 129:1723-1731; Yu et al. (2003) Phytochemistry 63:753-763; andU.S. Pat. Nos. 5,034,323; 5,283,184 and 5,942,657; each of which isincorporated herein by reference as if set forth in its entirety. Theefficiency of cosuppression may be increased by including a poly-dTregion in the expression cassette at a position 3′ to the sense sequenceand 5′ of the polyadenylation signal. See, US Patent ApplicationPublication No. 2002/0048814, incorporated herein by reference as if setforth in its entirety. Typically, such a nucleotide sequence hassubstantial sequence identity to the sequence of the transcript of theendogenous gene, preferably greater than about 65% sequence identity,more preferably greater than about 95% sequence identity, mostpreferably greater than about 99% sequence identity (e.g., U.S. Pat.Nos. 5;283,184 and 5,034,323; incorporated herein by reference as if setforth in its entirety).

Inhibition of the expression of the cytochrome P450 polypeptide of thepresent invention also can be obtained by antisense suppression. Forantisense suppression, the expression cassette is designed to express anRNA molecule complementary to all or part of a messenger RNA encodingthe cytochrome P450 polypeptide. Over-expression of the antisense RNAmolecule can result in reduced expression of the native gene.Accordingly, multiple plant lines transformed with the antisensesuppression expression cassette are screened to identify those that showthe greatest inhibition of nicotine demethylase polypeptide expression.

The polynucleotide for use in antisense suppression may correspond toall or part of the complement of the sequence encoding the cytochromeP450 polypeptide, all or part of the complement of the 5′ and/or 3′untranslated region of the cytochrome P450 polypeptide transcript, orall or part of the complement of both the coding sequence and theuntranslated regions of a transcript encoding the cytochrome P450polypeptide. In addition, the antisense polynucleotide may be fullycomplementary (i.e., 100% identical to the complement of the targetsequence) or partially complementary (i.e., less than 100% identical tothe complement of the target sequence) to the target sequence.

Antisense suppression also can be used to inhibit the expression ofmultiple proteins in the same plant (e.g., U.S. Pat. No. 5,942,657).Furthermore, portions of the antisense nucleotides may be used todisrupt the expression of the target gene. Generally, sequences of atleast 50 nucleic acids, 100, 200, 300, 400, 450, 500, 550 nucleic acids,or greater may be used. Methods for using antisense suppression toinhibit the expression of endogenous genes in plants are described,e.g., in Liu et al. (2002) Plant Physiol. 129:1732-1743; and U.S. Pat.Nos. 5,759,829 and 5,942,657; each of which is incorporated herein byreference as if set forth in its entirety. Efficiency of antisensesuppression may be increased by including a poly-dT region in theexpression cassette at a position 3′ to the antisense sequence and 5′ ofthe polyadenylation signal. See, US Patent Application Publication No.2002/0048814, incorporated herein by reference as if set forth in itsentirety.

For dsRNA interference, a sense RNA molecule like that described abovefor cosuppression and an antisense RNA molecule that is fully orpartially complementary to the sense RNA molecule are expressed in the,same cell, resulting in inhibition of the expression of thecorresponding endogenous messenger RNA. Expression of the sense andantisense molecules can be accomplished by designing the expressioncassette to comprise both a sense sequence and an antisense sequence forthe target cytochrome P450 sequence. Alternatively, separate expressioncassettes may be used for the sense and antisense sequences. Multipleplant lines transformed with the dsRNA interference expression cassetteor expression cassettes are then screened to identify plant lines thatshow the greatest inhibition of expression of the targeted cytochromeP450 polypeptide. Methods for using dsRNA interference to inhibit theexpression of endogenous plant genes are described in Waterhouse et al.(1998) Pros. Natl. Acad. Sci. USA 95:13939-13964; Liu et al. (2002)Plant Physiol. 129:1732′-1743; and International Patent ApplicationPublication Nos. WO 99/149024, WO 99/153050, WO 99/161631 and WO00/149035; each of which is incorporated herein by reference as if setforth in its entirety.

Amplicon expression cassettes comprise a plant virus-derived sequencethat contains all or part of the target gene but generally not all ofthe genes of the native virus. The viral sequences present in thetranscription product of the expression cassette allow the transcriptionproduct to direct its own replication. The transcripts produced by theamplicon may be either sense or antisense relative to the targetsequence (i.e., the messenger RNA for a cytochrome P450 polypeptide thatis involved in the metabolic conversion of nicotine to nornicotine).Methods of using amplicons to inhibit the expression of endogenous plantgenes are described, e.g., in Angell & Baulcombe (1997) EMBO J.16:3675-3684; Angell & Baulcombe (1999) Plant J. 20:357-362; and U.S.Pat. No. 6,646,805, each of which is incorporated herein by reference asif set forth in its entirety.

In some instances, the polynucleotide expressed by the expressioncassette of the invention is catalytic RNA or has ribozyme activityspecific for the messenger RNA of a cytochrome P450 polypeptidedescribed herein. Thus, the polynucleotide causes the degradation of theendogenous messenger RNA, resulting in reduced expression of thecytochrome P450 polypeptide. This method is described, for example, inU.S. Pat. No. 4,987,071, incorporated herein by reference as if setforth in its entirety.

In other instances, inhibition of the expression of one or more nicotinedemethylase polypeptides may be obtained by RNA interference byexpression of a gene encoding a micro RNA (miRNA). miRNAs are regulatoryagents consisting of about twenty-two ribonucleotides. miRNA are highlyefficient at inhibiting the expression of endogenous genes. See, e.g.,Javier et al. (2003) Nature 425:257-263, incorporated herein byreference as if set forth in its entirety.

For miRNA interference, the expression cassette is designed to expressan RNA molecule that is modeled on an endogenous miRNA gene. The miRNAgene encodes an RNA that forms a hairpin structure containing a22-nucleotide sequence that is complementary to another endogenous gene(target sequence). For suppression of nicotine demethylase polypeptideexpression, the 22-nucleotide sequence is selected from a cytochromeP450 polypeptide transcript sequence and contains 22 nucleotidesencoding this cytochrome P450 polypeptide sequence in sense orientationand 21 nucleotides of a corresponding antisense sequence that iscomplementary to the sense sequence. miRNA molecules are highlyefficient at inhibiting the expression of endogenous genes, and the RNAinterference they induce is inherited by subsequent generations ofplants.

Alternatively still, inhibition of the expression of one or morecytochrome P450 polypeptides may be obtained by hairpin RNA (hpRNA)interference or intron-containing hairpin RNA (ihpRNA) interference.These methods are highly efficient at inhibiting the expression ofendogenous genes. See, Waterhouse & Helliwell (20013) Nat. Rev. Genet.4:29-38 and the references cited therein.

The expression cassette for hpRNA interference may also be designed suchthat the sense sequence and the antisense sequence do not correspond toan endogenous RNA in this embodiment, the sense and antisense sequenceflank a loop sequence that comprises a nucleotide sequence correspondingto all or part of the endogenous messenger RNA of the target gene. Thus,it is the loop region that determines the specificity of the RNAinterference. See, e.g., International Patent Application PublicationNo. WO 02/00904, incorporated herein by reference as if set forth in itsentirety.

Transcriptional gene silencing (TGS) may be accomplished through use ofhpRNA constructs where the inverted repeat of the hairpin sharessequence identity with the promoter region of a gene to be silenced.Processing of the hpRNA into short RNAs that can interact with thehomologous promoter region may trigger degradation or methylation toresult in silencing (Aufsatz et al. (2002) Proc. Natl. Acad. Sci.99:16499-16506; and Mette et al. (2000) EMBO J. 19:5194-5201).

In some instances, the polynucleotide encodes an antibody that binds toat least one cytochrome P450 polypeptide, and reduces the activity of acytochrome P450 polypeptide of the present invention. In anotherembodiment, the binding of the antibody results in increased turnover ofthe antibody-cytochrome P450 polypeptide complex by cellular qualitycontrol mechanisms. The expression of antibodies in plant parts and theinhibition of molecular pathways by expression and binding of antibodiesto proteins in plant parts are well known in the art. See, e.g., Conrad& Sonnewald (2003) Nature Biotech. 21:35-36, incorporated herein byreference as if set forth in its entirety.

The activity of a cytochrome P450 polypeptide of the present inventioncan be reduced or eliminated by disrupting the gene encoding thecytochrome P450 polypeptide. The gene encoding the cytochrome P450polypeptide may be disrupted by any method known in the art, e.g., bytransposon tagging or by mutagenizing plants using random or targetedmutagenesis and selecting for plants that have reduced nicotinedemethylase activity or mutations in CYP82E4v2 or CYP82E5v2.

Transposon tagging may be used to reduce or eliminate the activity ofone or more cytochrome P450 polypeptides of the present invention.Transposon tagging comprises inserting a transposon within an endogenouscytochrome P450 gene to reduce or eliminate expression of the cytochromeP450 polypeptide.

Methods for the transposon tagging of specific genes in plants are wellknown in the art. See, e.g., Maes et al. (1999) Trends Plant Sci.4:90-96; Dharmapuri & Sonti (1999) FEMS Micerobiol. Lett. 179:53-59;Meissner et al. (2000) Plant J. 22:265-274; Phogat et al. (2000) J.Biosci. 25:57-63; Walbot (2000) Curr. Opin. Plant Biol. 2:103-107; Gaiet al. (2000) Nucleic Acids Res. 28:94-96; and Fitzmaurice et al. (1999)Genetics 153:1919-1928).

Additional methods for decreasing or eliminating the expression ofendogenous genes in plants are also known in the art and can besimilarly applied to the instant invention. These methods include otherforms of mutagenesis, using mutagenic or carcinogenic compoundsincluding ethyl methanesulfonate-induced mutagenesis, deletionmutagenesis, and fast neutron deletion mutagenesis used in a reversegenetics sense (with PCR) to identify plant lines in which theendogenous gene has been deleted. For examples of these methods see,Ohshima et al. (1998) Virology 213:472-481; Okubara et al. (1994)Genetics 137:867-874; and Quesada et al. (2000) Genetics 154:421-4315;each of which is incorporated herein by reference as if set forth in itsentirety. In addition, a fast and automatable method for screening forchemically induced mutations, TILLING (Targeting Induced Local LesionsIn Genomes), using denaturing HPLC or selective endonuclease digestionof selected PCR products is also applicable to the instant invention.See, McCallum et al. (2000) Nat. Biotechnol. 18:455-457, hereinincorporated by reference.

Mutations that impact gene expression or that interfere with thefunction of the encoded cytochrome P450 protein can be determined usingmethods that are well known in the art. Insertional mutations in geneexons usually result in null-mutants. Mutations in conserved residuescan be particularly effective in inhibiting the metabolic function ofthe encoded protein. Conserved residues of plant cytochrome P450polypeptides suitable for mutagenesis with the goal to eliminateactivity of a cytochrome P450 polypeptide in converting nicotine tonornicotine in a Nicotiana plant or plant part have been described.Conserved residues of plant cytochrome P450 polypeptides are disclosedin FIG. 1A-C, where the residues that differ from the other P450polypeptides are shaded in grey. The conserved residue is that which isnot shaded in grey at each position. Such mutants can be isolatedaccording to well-known procedures.

Dominant mutants can be used to trigger RNA silencing due to geneinversion and recombination of a duplicated gene locus. See, e.g.,Kusaba et al. (2003) Plant Cell 15:1455-1467.

While a number of sequences are recognized in the practice of theinvention, in particular sequences encoding SEQ ID NO:2 and 13 findparticular use. While not intending to be bound by any particularmechanism of action, the inventors believed that these sequences encodea nicotine demethylase that catalyzes the oxidative N-demethylation ofnicotine to nornicotine. Thus, methods to specifically inhibit thesecoding sequences and not other P450 sequences may be beneficial to therecombinant plant. That is, strategies that would lead to inhibition ofgene function of this individual locus may prove to be superior to thosethat inhibit the entire gene family. The P450 enzymes are involved inmany mechanisms in the plant, the inhibition of which may provedeleterious or detrimental to the growth and development of the plant ormay negatively impact factors such as the disease defense capabilitiesof the plant. Likewise, because the Nicotiana plant P450 enzymes havebeen implicated in plant metabolites such as phenylpropanoid, alkaloids,terpenoids, lipids, cyanogenic glycosides, glucosinolates, and a host ofother chemical entities, disruption of P450 activity may altercomponents involved in tobacco flavor, texture, or other properties thatwould impact the commercial usefulness of the plant. Therefore, the useof the methods discussed above to inhibit expression in a manner thatspecifically targets the sequence coding for SEQ ID NO:2 or 13 may bepreferred, including targeted mutational strategies, such aschimeraplasty. See, e.g., Stewart et al. (2000) Biotechniques24:838-843; and Graham et al. (2002) Biochim Biophys Acta 1587:1-6; eachof which is incorporated herein by reference as if set forth in itsentirety. In some embodiments, the methods discussed above are used toinhibit expression in a manner that specifically targets SEQ ID NO:1(encoding SEQ ID NO:2), SEQ ID NO:50 (encoding SEQ ID NO:13), or both ofthese sequences.

The compositions of the invention can be used in screening methods toidentify nonconverter plants for use in breeding programs. In thismanner, the nucleotide sequences of the invention can be used to screennative germplasms for nonconverter plants having a stable mutation inone or more P450 genes identified herein. These nonconverter plantsidentified by the methods of the invention can be used to developbreeding lines.

In addition to the nucleotide sequences encoding P450 coding sequences,compositions of the invention include an intron sequence in theCYP82E5v2 sequence that can be used in screening methods. While notintending to be bound by any particular mechanism of action, theCYP82E5v2 gene(s) may represent the only member(s) of the cytochromeP450 family involved in the metabolic conversion of nicotine tonornicotine (and as stated previously there is a good likelihood thatthe CYP82E5v2 cDNAs originated from a single unique genetic locus) ingreen-leaves of tobacco. For certain applications, it would be useful tohave a means of diagnostically differentiating this specific member ofthe cytochrome P450 gene family from the rest of the closely relatedsequences within this family. For example, it is possible that withinthe naturally existing tobacco germplasm (or in mutagenizedpopulations), accessions may exist in which this gene is naturallydysfunctional and may therefore may be valuable as a permanentlynonconverter resource. Such dysfunctional genes may include thoseencoding the polypeptides set forth in SEQ ID NOS:6 and 12. A method tospecifically assay for such genotypes (e.g., deletion mutants,rearrangements, and the like) could serve as a powerful tool. Thepresent invention includes primers designed to specifically amplify exon1 and exon 2 of CYP82E5v2 or CYP82E4v2 where one of the two primer pairscorresponds to the intron between the exons. Examples of primers usefulto amplify the exons of CYP82E4v2 include SEQ ID NO:32 with SEQ IDNOS:33-35. Examples of primers useful to amplify the exons of CYP82E5v2include SEQ ID NO:36 with 37 and SEQ ID NO:38 with 39. These sameprimers can be used for sequence analysis of the products.

When any cDNA corresponding to a member of the CYP82E2 family is used asa hybridization probe in a Southern blotting assay of tobacco genome isDNA, a complex pattern is observed. This is expected, given that thereare multiple, closely related members of this gene family. Because theintron regions of genes are typically less conserved than exons, it ispredicted that the use of an intron-specific probe would reduce thiscomplexity and better enable one to distinguish the gene(s)corresponding to the CYP82E4v2 gene or the CYP82E5v2 gene from the othermembers of the family. The use of a CYP82E4v2 or CYP82E5v2intron-specific probe, and/or the PCR primers used to generate productsprovide powerful tools in assays to determine whether any naturallyoccurring, or mutagenized, tobacco plants possess deletions orrearrangements that may render the gene inactive. Such a plant can thenbe used in breeding programs to create tobacco lines that are incapableof converting.

Transformed Plants, Plant Parts and Products Having Reduced Nornicotineand NNN Content

The cytochrome P450 polynucleotides of the invention, and variants andfragments thereof, can be used in the methods of the present inventionto inhibit cvtochrome P450s that are involved in the metabolicconversion of nicotine to nornicotine in a plant. In this manner,inhibitory sequences that target expression or function of a cytochromeP450 polypeptide disclosed herein are introduced into a plant or plantcell of interest. The expression cassettes described herein can beintroduced into a plant of interest, for example, a Nicotiana plant asnoted herein below, using any suitable transformation methods known inthe art including those described herein.

The methods of the invention do not depend on a particular method forintroducing a sequence into a plant or plant part, only that the desiredsequence gains access to the interior of at least one cell of the plantor plant part. Methods for introducing polynucleotide sequences intoplants are known in the art and include, but are not limited to, stabletransformation methods, transient transformation methods andviral-mediated transformation methods.

Transformation protocols as well as protocols for introducingheterologous polynucleotide sequences into plants vary depending on thetype of plant or plant cell targeted for transformation. Suitablemethods of introducing polynucleotides into plant cells of the presentinvention include microinjection (Crossway et al. (1986) Biotechniques4:320-334), electroporation (Shillito et al. (1987) Meth. Enzymol.153:313-336; and Riggs et al. (1986) Proc. Natl. Acad Sci. USA83:5602-56116), Agrobacterium-mediated transformation (U.S. Pat. Nos.5,104,310; 5,149,645; 5,177,010; 5,231,019; 5,463,174; 5,464,763;5,469,976; 4,762,785; 5,004,863; 5,159,135; 5,563,055 and 5,981,840),direct gene transfer (Paszkowski et al. (1984) EMBO J. 1:2717-2722) andballistic particle acceleration (U.S. Pat. Nos. 4,945,050; 5,141,131;5,886,244; 5,879,918 and 5,932,782; Tomes et al. (1995) in Plant cell,tissue, and organ culture fundamental methods, ed. Gamborg & Phillips(Springer-Verlag, Berlin); McCabe et al. (1988) Biotechnology6:923-926). See also, Weissinger et al. (1988) Ann. Rev. Genet.22:421-477; Christou et al. (1988) Plant Physiol. 87:671-674 (soybean);McCabe et al. (1988) Bio/Technology 6:923-926 (soybean); Finer &McMullen (1991) In Vitro Cell Dev. Biol. 27P:175-182 (soybean); Singh etal. (1998) Theor. Appl. Genet. 96:319-324 (soybean); De Wet et al.(1985) in The experimental manipulation of ovule tissues, ed. Chapman etal. (Longman, New York.); pp. 197-209 (pollen); Kaeppler et al. (1990)Plant Cell Reports 9:415-418; and Kaeppler et al. (1992) Theor. Appl.Genet, 84:560-566 (whisker-mediated transformation); D'Halluin et al.(1992) Plant Cell 4:1495-1505 (electroporation); each of which isincorporated herein by reference as if set forth in its entirety.

Any plant tissue that can be subsequently propagated using clonalmethods whether by organogenesis or embryogenesis, may be transformedwith a recombinant construct comprising a cytochrome P450 inhibitorysequence, for example, an expression cassette of the present invention.As used herein, “organogenesis” means a process by which shoots androots are developed sequentially from meristematic centers. As usedherein, “embryogenesis” means a process by which shoots and rootsdevelop together in a concerted fashion (not sequentially), whether fromsomatic cells or gametes. Exemplary tissues that are suitable forvarious transformation protocols described herein include, but are notlimited to, callus tissue, existing meristematic tissue (e.g., apicalmeristems, axillary buds and root ineristems) and induced meristemtissue (e.g., cotyledon meristem and hypocotyl meristem), hypocotyls,cotyledons, leaf disks, pollen, embryos and the like.

As used herein, “stable transformation” means that the nucleotideconstruct of interest introduced into a plant integrates into the genomeof the plant and is capable of being inherited by the progeny thereof.Likewise, “transient transformation” means that a sequence is introducedinto the plant and is only temporally expressed or is only transientlypresent in the plant.

The inhibitory sequences of the invention can be provided to a plantusing a variety of transient transformation methods. The inhibitorysequences of the invention can be transiently transformed into the plantusing techniques known in the art. Such techniques include viral vectorsystems and the precipitation of the polynucleotide in a manner thatprecludes subsequent release of the DNA. Thus, the transcription fromthe particle-bound DNA can occur, but the frequency with which it isreleased to become integrated into the genome is greatly reduced. Suchmethods include the use of particles coated with polyethyenlimine (PEI;Sigma 4P3143).

Alternatively, the inhibitory sequence of the invention may beintroduced into plants by contacting plants with a virus or viralnucleic acids. Generally, such methods involve incorporating anexpression cassette of the invention within a viral DNA or RNA molecule.Promoters for use in the expression cassettes of the invention alsoencompass promoters utilized for transcription by viral RNA polymerases.Methods for introducing polynucleotides into plants and expressing aprotein encoded therein, involving viral DNA or RNA molecules, are knownin the art. See, e.g., U.S. Pat. Nos. 5,889,191; 5,889,190; 5,866,785;5,589,367; 5,316,931; and Porta et al. (1996) Molecular Biotechnology5:209-221; each of which is incorporated herein by reference as if setforth in its entirety.

Transformed cells may be grown into Nicotiana plants in accordance withconventional methods. See, e.g., Vasil & Hildebrandt (1965) Science150:889; Negaard & Hoffman (1989) Biotechniques 7(8):808-812. Theseplants may then be grown, and either pollinated with the sametransformed line or different lines, and the resulting progeny havingexpression of the desired phenotypic characteristic identified, i.e.,reduced expression of one or more nicotine demethylases that areinvolved in the metabolic conversion of nicotine to nornicotine, andthus reduced content of nornicotine, and a concomitant reduced contentof its nitrosamine metabolite, NNN, in the plant, particularly in theleaf tissues. Two or more generations may be grown to ensure thatexpression of the desired phenotypic characteristic is stably maintainedand inherited and then seeds harvested to ensure expression of thedesired phenotypic characteristic has been achieved. In this manner, thepresent invention provides transformed seed (also referred to as“transgenic seed”) having a polynucleotide of the invention, forexample, an expression cassette of the invention, stably incorporatedinto their genome.

The compositions and methods of the invention can be used to reduce thenornicotine content, particularly in the leaves and stems, of any plantof the genus Nicotiana including, but not limited to, the followingspecies: acuminata, affinis, alata, attenuate, bigelovii, clevelandii,excelsior, forgetiana, glauca, glutinosa, langsdorffii, longiflora,obtusifolia, palmeri, paniculata, plumbaginifolia, qudrivalvis, repanda,rustica, suaveolens, sylvestris, tabacum, tomentosa, trigonophylla and xsanderae. The present invention also encompasses the transformation ofany varieties of a plant of the genus Nicotiana, including, but notlimited to, Nicotiana acuminata multiflora, Nicotiana alata grandiflora,Nicotiana bigelovii quadrivalvis, Nicotiana bigelovii wallacei,Nicotiana obtusifolia obtusifolia, Nicotiana obtusifolia plameri,Nicotiana quadrivalvis bigelovii, Nicotiana quadrivalvis quadrivalvis,Nicotiana quadrivalvis wallacei, and Nicotiana trigonophylla palmeri, aswell as varieties commonly known as flue or bright varieties, Burleyvarieties, dark varieties and oriental/Turkish varieties.

The transgenic plants of the genus Nicotiana as described herein aresuitable for conventional growing and harvesting techniques, such ascultivation in manure rich soil or without manure, bagging the flowersor no bagging, or topping or no topping. The harvested leaves and stemsmay be used in any traditional tobacco product including, but notlimited to, pipe, cigar and cigarette tobacco, and chewing tobacco inany form including leaf tobacco, shredded tobacco, or cut tobacco.

Thus the present invention provides a Nicotiana plant, particularly leaftissues of these plants, comprising an expression cassette of theinvention and a reduced amount of nornicotine and N′-nitrosonornicotine.As used herein, “a reduced amount” or “a reduced level” means an amountof nornicotine and/or NNN in a treated or transgenic plant of the genusNicotiana or a plant part or tobacco product thereof that is less thanwhat would be found in a plant of the genus Nicotiana or a plant part ortobacco product from the same variety of tobacco, processed (i.e.,cultured and harvested) in the same manner, that has not been treated orwas not made transgenic for reduced nornicotine and/or NNN. The amountof nornicotine may be reduced by about 10% to greater than about 90%,including greater than about 20%, about 30%, about 40%, about 50%, about60%, about 70% and about 80%. The conversion of nicotine to nornicotinecan be less than 0.3%, less than 0.5%, less than 0.7%, between0.1%-0.5%, between 0.1%-0.4%, between 0.1%-0.7%, or between 0.1%-1.0% inplants, plant parts, and products of the present invention, and morespecifically in plants, plant parts having mutations in CYP82E4v2 andCYP825v2.

As used herein, “tobacco products” means, but is not limited to, smokingmaterials (e.g., any cigarette, including a cigarillo, a non-ventilatedor vented recess filter cigarette, a cigar, pipe tobacco), smokelessproducts (e.g., snuff, chewing tobacco, biodegradable inserts (e.g.,gum, lozenges, dissolving strips)). See, e.g., US Patent ApplicationPublication No. 2005/0019448, incorporated herein by reference as if setforth in its entirety. Tobacco product also includes blends that can bemade by combining conventional tobacco with differing amounts of the lownornicotine and/or NNN tobacco described herein. Tobacco products alsoincludes plant or plant part of the genus Nicotiana as described aboveis cured tobacco. The tobacco products described herein reduce thecarcinogenic potential of tobacco smoke that is inhaled directly withconsumption of a tobacco product such as cigars, cigarettes, or pipetobacco or inhaled as secondary smoke (i.e., by an individual thatinhales the tobacco smoke generated by an individual consuming a tobaccoproduct such as cigars, cigarettes, or pipe tobacco). The cured tobaccodescribed herein can be used to prepare a tobacco product, particularlyone that undergoes chemical changes due to heat, comprising a reducedamount of nornicotine and/or NNN in the smoke stream that is inhaleddirectly or inhaled as secondary smoke. In the same manner, the tobaccoproducts of the invention may be useful in the preparation of smokelesstobacco products such as chewing tobacco, snuff and the like.

The tobacco products derived from the transgenic tobacco plants of thepresent invention thus find use in methods for reducing the carcinogenicpotential of these tobacco products, and reducing the exposure of humansto the carcinogenic nitrosamine, NNN, particularly for individuals thatare users of these tobacco products. The following examples are offeredby way of illustration and not by way of limitation.

EXPERIMENTAL

The following materials and protocols are utilized in the experimentsdescribed herein below.

Plant Materials

All plant materials were obtained from Dr. Earl Wernsman, Department ofCrop Science, North Carolina State University (Raleigh, N.C.). N.tomentosiformis and N. sylvestris seed were obtained from the Nicotianagermplasm collection maintained by North Carolina State University.Plants were grown in growth chambers for two months until they weretransferred to a greenhouse. Burley lines DH 98-325-5 (nonconverter) andDH 98-325-6 (converter) represent near-isogenic lines, i.e., recoveredfrom the same maternal haploid plant. SC58 is a flue-cured tobaccovariety, nonconverter individuals of which are designatedSC58(c_(T)c_(T)). SC58(C_(T)C_(T)) is a near-isogenic stable converterline that originated though the introgression of the single dominantconverter locus (C_(r)) found in the tobacco progenitor species N.tomentosiformis into SC58 (Mann et al. (1964) Crop Sci., 4:349-353).After eight additional backcrosses to SC58, the near-isogenicSC58(C_(T)C_(T)) line was created and was subsequently maintained viaself-fertilization.

All plants were maintained in growth chambers or greenhouses usingstandard potting soil and fertilizer. Plants kept in a 14/10 light/darkcycle, were watered once a day as needed and fertilized with PetersProfessional® All Purpose Plant Food fertilizer (20-20-20; SpectrumBrands Inc.; Madison, Wis.) once a week. To facilitate senescence,detached green leaves were treated by dipping each leaf for 30 secondsin a 0.2% ethephon solution. Leaves were cured in plastic bags underdark conditions until they turned yellow.

For the ethyl methane sulphonate (EMS) mutagensis, two grams of seedfrom the strong converter Burley tobacco line DH98-325-6 weresurface-sterilized in 50% bleach for 12 minutes and rinsed 6 times insterile dH₂O. Approximately 80 mg aliquots were then placed in screw capvials and imbibed in 1 ml dH₂O for 12 hours. The dH₂O was decanted, andthe seed was incubated in 1 ml of 0.5% EMS (Sigma; St. Louis, Mo.) androcked gently on a Nutator° Mixer (TCS Scientific Corp.; New Hope, Pa.)at room temperature for 12 hours in the dark. After removal of the EMSsolution, the treated seeds were washed eight times in 1 ml volumes ofdH2O with gentle shaking for 5 minutes. After the final wash, seeds werecollected onto a Buchner funnel for a final rinse and subsequentlyallowed to dry on filter paper before sowing on soil. Seedlings weregrown on standard float trays for about 7 weeks, then transplanted to afield. Field-grown plants were allowed to self-pollinate, and 5-10capsules (containing M₁ generation seed) per plant were collected andcatalogued corresponding to approximately 4000 independent M₀individuals.

Isolation of NtabCYP82E5v2 cDNA

Total RNA was extracted from green and senescing leaves of DH98-325-6tobacco using TRIzol® (Invitrogen; Carlsbad, Calif.) following theprotocol of the manufacturer. Genomic DNA was removed by treating theextracts with TURBO DNA-free™ DNase Kit (Ambion; Austin, Tex.) followingthe protocol of the manufacturer. cDNA synthesis was performed with theStrataScript™ First-Strand Kit (Stratagene; Cedar Creek, Tex.) usingoligo dT primers and 3 μg of DNase-free total RNA. Full-lengthNtabCYP82E5v2 cDNAs were amplified using the E5Orf_F forward and E5Orf_Rreverse primers, 10 ng tobacco leaf cDNA library as a template and LongRange™ PCR Enzyme Blend (GeneChoice, Inc.; Frederick, Md.) following theprotocol of the manufacturer. DNA sequence information of the primers islisted in the sequence listing and above. PCR conditions were asfollows: 3 minutes initial denaturation at 95° C. followed by 33 cyclesof denaturation at 94° C. for 30 seconds, annealing at 60° C., for 30seconds and extension at 68° C. for 90 seconds, followed by anincubation at 68° C. for 7 minutes. PCR products were cloned intopGEM®-T Easy cloning vector (Promega Corp.; Madison, Wis.) and sequencedaccording to the dideoxy method using synthetic oligonucleotides asprimers (Sanger et al. (1977) Proc. Natl. Acad. Sci. U. S. A. 74:5463-5467).

Isolation of the Genomic CYP82E5 Fragments

Genomic DNA was isolated from the green leaves of N. tomentosiformis andDH98-325-6 tobacco using a cetyltrimethyl ammonium bromide-basedextraction procedure according to the instructions of the NucleoSpin®Plant kit (BD Bioscience Clontech; Palo Alto, Calif.).

Genomic fragments of the CYP82E5 orthologs were amplified in twoconsecutive PCR reactions using nested primers. The first PCR mixturecontained 0.5 μM each of the E5Gen_F1 forward (SEQ ID NO:40) andE5Gen_R1 reverse (SEQ ID NO:41) primers, 200 μM of each dNTP, 20 ng ofgenomic DNA template and 2 U of Platinum® Taq polymerase (Invitrogen).PCR amplification was performed under the following conditions: 90° C.for 3 minutes; 30 cycles of denaturation at 94° C. for 30 seconds,annealing at 57° C. for 30 seconds and extension at 68° C. for 2.2minutes followed by a final extension at 68° C. for 5 minutes. Thecomposition and conditions of the second PCR were the same as thosedescribed for the first amplification, except primers E5Gen F2 (SEQ IDNO:42) and E5Gen_R2 (SEQ ID NO:43) were added and 1 μl of a 50-folddiluted sample of the first PCR product was used as a template. Thepurified products of the final PCR were ligated into the pGEM-T-Easycloning vector and subjected to DNA sequencing.

Expression of the NtabCYP82E5v2 cDNA in Yeast

To facilitate the expression of the NtabCYP82E5v2 cDNA in yeast, theopen reading frame of the gene was inserted downstream of thegalactose-inducible, glucose-repressed GAL10-CYC1 promoter of thepYeDP60 yeast expression vector (Cullin & Pompon 1988). The recombinantplasmid was introduced into WAT11 yeast strain using thelithium-acetate-based transformation method as described by Gietz et al.(1992) Nucleic Acids Res. 20:1425-1425. Yeast culturing,galactose-mediated induction of gene expression and the isolation of themicrosomal fractions were performed according to the protocol of Pomponet al. (1996) Methods Enzymol. 272:51-64.

Kinetic Analysis of NtabCYP82E5-Mediated Nicotine Metabolism

The nicotine metabolism assays were performed in a reaction mixturecontaining 0.75 mM NADPH, 2.5 μM [pyrrolidine-2-¹⁴C] nicotine (MoravekBiochemicals; Brea, Calif.) and 90 μg of yeast microsomes in a finalvolume of 150 μl Pi buffer, pH 7.1. Nicotine concentrations wereadjusted by the addition of nonradiolabelled nicotine to finalconcentrations ranging between 0.7 and 13 μM. Microsomal preparationsisolated from yeast containing an empty vector were used as negativecontrol. Reaction mixtures were incubated at 25° C. for 7 minutes, andthe reaction was arrested with 50 μl of acetone. After centrifugation at16,000×g for 3 minutes, 50 μl the sample was spotted on a 250-μm WhatmanK6F silica plate. The plates were developed incholoroform:methanol:ammonia (60:10:1) solvent system. Nicotine and itsnornicotine derivative were quantified by measuring the relativeabundance of their radioactive traces with a Bioscan System 400 imagingscanner. K_(m) and v_(max) values were calculated by fitting theMichaelis-Menten equation to nicotine demethylation measurements atdifferent substrate concentrations using the SigmaPlot® 10.0 graphingsoftware (Systat Software, Inc.; San Jose, Calif.).

Quantitative Real-Time PCR Analysis of CYP82E4v2 and CYP82E5v2Expression Expression of the N. tomentosiformis and tobacco orthologs ofCYP82E4v2 and CYP82E5v2 were analyzed by allele-specific, quantitativereal-time PCR (qrt-PCR) analysis using SYBR® Green I fluorescencechemistry (Morrison et al. (1998) Biotechniques 24: 954-962). Total RNAwas extracted from the green and cured leaves of 3-month-old plantsusing the methods already described for NtabCYP82E5v2 cDNA isolationherein. CYP82E4v2 and CYP82E5v2 transcripts were amplified using theE4Rt_F (SEQ ID NO:44) and E5Rt_F (SEQ ID NO:46) forward in conjunctionwith the E4Rt_R (SEQ ID NO:45) and the E5Rt_R reverse (SEQ ID NO:47)primers, respectively. The qrt-PCR mixture contained 1× iQ™ SYBR® GreenSupermix (Bio-Rad Laboratories) 0.5 μM of each primer and 0.5 μg ofcDNA. Transcript measurements were obtained using standard curvesgenerated by amplifying known amounts of target DNA and a 220-bpfragment of the glyceraldehyde-3-phosphate dehydrogenase (G3PDH) gene,amplified with the G3PDH_F (SEQ ID NO:48) and G3PDH_R (SEQ ID NO:49)primers, to provide an internal standard for cDNA measurements. DNAamplifications were performed using the real time PCR system (Bio-RadLaboratories; Hercules, Calif.) under the following conditions: 95° C.for 2 minutes; 35 cycles of 95° C. for 30 seconds, 57 C for 30 seconds,72° C. for 50 seconds followed by final extension at 72° C. for 5minutes. DNA sequences of the amplicons generated by qrt-PCR wereverified by cloning the PCR products and sequencing 20 randomly selectedclones.

Transgenic Plant Analysis

The RNAi-based gene silencing constructs are assembled in a version ofthe pKYL80 cloning vector (Schardl et al. (1987) Gene 61:1-11) that isengineered to contain a 151-bp fragment of the soybean FAD3 gene intronbetween the Xhol and Sacl restriction sites of the polylinker(pKYLX80I). To create a construct in which the FAD3 intron was flankedby a sense and antisense fragment of CYP82E5v2, a 400-bp intron regionis cloned between the HindIII-Xhol and Sacl-Xbal restriction sites ofpKYLX801 in its sense and antisense orientation, respectively. Theresulting HindIII-Xbal fragment containing the CYP8E5v2 sense arm, FAD3intron, and CYP82E5v2 antisense arm is subcloned into the pKYLX71 plantbinary expression vector (Maiti et al. (1993) Proc. Natl. Acad. Sci. USA90:6110-6114) between the 35S CaMV promoter and a rubisco small subunitterminator.

Overexpression constructs are created by replacing the 3-glucuronidaseORF of the plant binary expression vector pBI121 (Clontech) with thefull-length coding regions of the CYP82E5v2, CYP82E5v2 variants, orCYP82E4v2 variants cDNAs. This places the tobacco P450s under thetranscriptional control of the 35S CaMV promoter. The pBI121- andpKYLX71-based constructs are transformed into Agrobacterium tumefaciensstrain LBA 4404 and introduced into tobacco cultivars Petite Havana andDH98-325-6 (converter), respectively, using established protocols(Horsch et al. (1985) Science 227:1229-1231).

Northern Blot Analysis

Total cellular RNAs are isolated from tobacco leaves using the TRIZOL®method as described by the manufacturer (Invitrogen). Five to tenmicrograms of RNA are size fractionated on a 1.2% agarose gel preparedin TBE buffer. RNA immobilization, probe labeling, and signal detectionare carried out using the DIG nucleic acid labeling and detection kitsaccording to the manufacturer's instructions (Roche). Alternatively,probes are synthesized using ³²P-dCTP according to protocolsaccompanying the Random Primed DNA Labeling kit (Roche).

Isolation of DNA from EMS-Mutagenized Plants.

Genomic DNA was isolated from young leaf material of a singlegreenhouse-grown M₁ plant from each independent M₁ seed pool. 1.5 mlscrew cap vials were used to collect a single leaf disc from each plantand the samples were stored at −80° C. until they were processed. Theleaf tissue was deposited to the bottom of the vial by centrifugation,and the tubes were set in a shallow amount of liquid nitrogen tofacilitate grinding using plastic pestles. Genomic DNA was isolated fromthe ground material by adding 320 μl extraction buffer (200 mM Tris-Cl,pH 7.5, 250 mM NaCl, 25 mM EDTA, 0.5% SDS). Samples were vortexed for 20seconds, and left at room temperature for 5 minutes, followed by a 37°C. incubation for 3 minutes. The lysed material was centrifuged 10minutes, and the supernatant transferred to fresh tube. 50 μl of proteinprecipitation solution (Qiagen) was added, the samples briefly vortexed,then set on ice for 5 minutes, followed by a 4 minutes centrifugation at16,000×g. Precipitation of nucleic acids was accomplished by adding 0.7volume isopropanol to the supernatant followed by centrifugation for 7minutes at 12,000×g. After a 70% ethanol wash, the pellet wasresuspended in 50 μl TE.

Template Preparation for Sequencing.

CYP82E4v2 and CYP82E5v2 each contain two exons separated by a largeintron (1001 bp and 1054 bp, respectively). PCR primers are designed tospecifically amplify exon 1 and exon 2 for each gene. Due to the highsequence homology shared among the cDNAs of CYP82E2 gene family members,two primer pairs correspond to intron sequence in order to obtainamplification products specific to the desired gene (owing to the factthat the intron sequences of this gene family are not nearly asconserved as the exon sequences). The exon 1-specific primers forCYP82E4v2 are 5′-TGGAATTATGCCCATCCTACA-3′ (forward) (SEQ ID NO:32) and5′-CATTAGTGGTTGCACCTGAGG-3′ (reverse) (SEQ ID NO:33). CYP82E4v2 exon2-specific primers are 5′-GATGAGATGTGTGCATACTTG-3′ (forward) (SEQ ID NO:34) and 5′-CCAAATTAGAAAAACTCGTACTG-3′ (reverse) (SEQ ID NO:35). ForCYP82E5v2, the primers specific for amplifying exon 1 are5′-ATTGTAGGACTAGTAACCCTTACAC-3′ (forward) (SEQ ID NO:36) and5′-GAGGCACAAAGAATTCTCATC-3′ (reverse) (SEQ ID NO:37); and for CYP82E5v2exon 2, the primers 5′-GAGTAGAGGGATTGTTTCCG-3′ (forward) (SEQ ID NO:38)and 5′-GTACAATCAAGATAAAACATCTAAGG-3′ (reverse) (SEQ ID NO:39) are used.PCR reactions were conducted in 96-well microtiter plates using 1 μl oftemplate in a 25 μl reaction volume containing 10 μmoles each primer,200 μM dNTP, 1.5 mM MgCl and 1.4 units of high fidelity Taq DNApolymerase (Roche). DNA amplification of CYP82E4v2 sequences wasperformed using an initial 3 minutes denaturation at 94° C., followed by30 cycles of denaturation at 94° C. for 30 seconds, primer annealing at55° C. for 30 seconds, and extension at 72° C. for 1.5 minutes, followedby a final 7 minutes 72° C. extension. Amplification conditions forCYP82E5v2 gene fragments were the same except the annealing temperaturewas 53° C. PCR reaction products were purified using Millipore's MontagePCμ96 filter units. Agarose gel electrophoresis was used to estimate thequantity and quality of the cleaned up PCR products prior to sequencing.

Sequence Analysis.

The PCR products were subjected to cycle sequencing using AppliedBiosystems Big Dye Version 3.1 in 96-well format sequencing reactionsaccording to the manufacturer's instructions. The primers used forsequencing (3.2 μmoles per reaction) are as follows:5′-TGGAATTATGCCCATCCTACA-3′ (SEQ ID NO:32) and5′-CCTATAAAAAGGAAGTTGCCG-3′ (SEQ ID NO:44) (forward and reverse primersfor exon 1 of CYP82E4v2); 5′-GATGAGATGTGTGCATACTTG-3′ (SEQ ID NO:34) and5′-CCAAATTAGAAAAACTCGTACTG-3′ (SEQ ID NO:35) (forward and reverseprimers for exon 2 of CYP82E4v2); 5′-ATTGTAGGACTAGTAACCCTTACAC-3′ (SEQID NO:36) and 5′-CTCATCTTTTTTCCATTTATCATTC-3′ (SEQ ID NO:45) (forwardand reverse primers for exon 1 of CYP82E5v2); and5′-CAAGGTTCGGCAGATAGTG-3′ (SEQ ID NO:46) and5′-GTACAATCAAGATAAAACATCTAAGG-3′ (SEQ ID NO:39) (forward and reverseprimers for exon 2 of CYP82E5v2). Sequencing reactions were cleaned upusing EdgeBioSystems 96-well plates and analyzed using high-throughputApplied Biosystems 3700 or 3730 capillary sequencers. Sequences werealigned using the Clustal W algorithm as represented in the Vector NTIsoftware package (Invitrogen). Putative mutations are verified byrepeating the sequence analysis on sibling M₁ plants grown from thecognate M₀ seed lot.

Alkaloid Analysis

Alkaloid analyses are performed by gas chromatography as describedpreviously. Briefly, alkaloid analyses are performed by detachingtobacco leaves and dipping them in a solution of 1% ethephon, then,air-curing in a growth chamber for 7-10 days. Cured leaves are dried at50° C. for two days and ground to a fine powder. A quantitativedetermination of the nicotine, nornicotine, anatabine and anabasinecontent was made using a Perkin Elmer Autosystem XL Gas Chromatographaccording to the “LC-Protocol” established at the University of Kentucky(available online at the University of Kentucky's website), herebyincorporated by reference.

Example 1: Isolation and Characterization of CYP82E5V2

Nicotine Conversion in N. tomentosiformis and Various Tobacco Genotypes

To assess the alkaloid composition of wild-type plants, concentrationsof nicotine and nornicotine were determined in the green and curedleaves of N. tomentosiformis and various tobacco genotypes using gaschromatography. In N. tomentosiformis, nornicotine appears as thepredominant alkaloid in both the green and cured leaves (Table 1).Converter Burley cultivar DH-98-325-6 and converter Flue-Cured cultivarSC58C contain low levels of nornicotine (about 3%) in the green leaves,but a large percentage (about 95 and 25%, respectively) nicotine contentis metabolized into nornicotine once the leaves senesce (see, Table 1).Levels of nornicotine are low in both the green and cured leaves ofnonconverter Burley and Flue-Cured cultivars DH98-325-5 and SC58NC,respectively, although nicotine conversion slightly increases aftercuring.

TABLE 1 Alkaloid analysis of green and cured leaves of N.tomentosiformis and various tobacco genotypes. % Nicotine^(a) %Nornicotine^(a) % Conversion^(b) Plant Phenotype Green Cured^(c) GreenCured^(c) Green Cured N. tomentosiformis GLC 0.003^(d) 0.007 0.188 0.29998.4 97.7 (0.0002) (0.001) (0.022) (0.031) (2.4) (2.1) DH98-325-5 NC1.243 1.332 0.032 0.040 2.6 3.0 (0.334) (0.321) (0.006) (0.008) (0.2)(0.3) DH98-325-6 SLC 1.113 0.082 0.041 1.416 3.5 94.4 (0.095) (0.019)(0.004) (0.187) (0.1) (1.6) SC58NC NC 1.422 1.487 0.032 0.039 2.2 2.5(0.127) (0.089) (0.004) (0.001) (0.07) (0.1) SC58C SLC 1.171 0.501 0.0290.164 2.4 24.7 ^(a)Percentage of leaf dry weight. ^(b)[% nornicotine (%nicotine + % nornicotine)⁻¹] × 100. ^(c)Leaves are treated with 0.2%ethephon and cured until they turned yellow. ^(d)Mean over the alkaloidcontent of three plants, except SC58C where one plant was used due tothe low frequency of conversion. ^(e)Values in parenthesis representstandard error of the mean. Abbreviations: GLC, green-leaf converter;NC, nonconverter; SLC, senescing-leaf converter.Isolation of the NtabCYP82E5v2 cDNA from Tobacco

To identify putative nicotine demethylase genes from tobacco, aPCR-based gene amplification strategy was used where the primers arecomplementary to the 5′ and 3′ termini of the NtabCYP82E3 coding region.Primer design was based on the observation that all members of theclosely-related CYP82E2 gene subfamily display a high degree of DNAsequence identity at the regions immediately following the ATGinitiation signal and preceding the TAA stop codon (Siminszky et al.(2005) Proc. Natl. Acad. Sci. U. S. A. 102: 14919-14924) providing wellconserved primer target sites for the amplification of additional familymembers. A PCR using CYP82E3-specific primers and permissive annealingtemperatures was conducted to amplify putative nicotine demethylasegenes from a tobacco green leaf cDNA library. DNA sequence analysis of20 randomly selected clones generated from the PCR products yielded 19NtabCYP82E3 cDNA sequences and one cDNA whose DNA sequence is differentfrom all functionally characterized members of the CYP82E2 genesubfamily (Siminszky et al. (2005) Proc. Natl. Acad. Sci. U. S. A. 102:14919-14924). A homology search against the GenBank database using theBLAST algorithm reveals that the clone representing the unique DNAsequence shares a 99.7% nucleotide and a 99.2% predicted amino acididentity with the tobacco CYP82E5v1 cDNA of unknown function. Accordingto the guidelines of the P450 nomenclature committee, the cDNA was namedNtabCYP82E5v2. The predicted amino acid sequence of NtabCYP82E5v2 is89.2, 91.9 and 91.3% identical to that of NtabCYP82E2 , NtabCYP82E3 andNtabCYP82E4v2 , respectively.

NtabCYP82E5v2 Encodes for a Functional Nicotine Demethylase

To determine whether NtabCYP82E5v2 encodes nicotine demethylaseactivity, the N.tabCYP82E5 cDNA is expressed in the WAT11 yeast strain.The WAT11 strain was engineered to provide high efficiency transgeneexpression and an optimal redox environment for P450-mediated reactionsby expressing the Arabidopsis P450 reductase gene, ATR1, under thetranscriptional control of a galactose-inducible promoter (Pompon et al.(1996) Methods Enzymol. 272: 51-64). Microsomal fragments isolated fromyeast expressing NtabCYP82E5v2 actively catalyze the N-demethylation ofnicotine. The K_(m) and v_(max) values for the NtabCYP82E5v2-mediatednicotine demethylase reaction are 5.6 ±1.4 μM and 0.7±0.02 nmol min⁻¹mg⁻¹ protein (mean±standard error), respectively. In contrast, nonicotine demethylase activity is evident when microsomes isolated fromyeast transformed with an empty plasmid are added to the catalyticassay.

NtabCYP82E5v2 is Derived from Progenitor N. tomentosiformis

As the allotetraploid genome of tobacco consists of two geneticcomponents, the S genome donated by N. sylvestris and the T genomederived from N. tomentosiformis, which of the two progenitor speciescontributed NtabCYP82E5v2 to tobacco is unknown.

To this end, CYP82E5-specific primers were designed to amplify a genomicfragment of the CYP82E5 genes from tobacco and theNtabCYP82E5v2-donating progenitor. PCR amplifications produce a distinct2243 bp product when genomic DNA isolated from tobacco or N.tomentosiformis is used as template, but amplicons are not detected whengenomic DNA of N. sylvestris is amplified indicating that NtabCYP82E5v2was derived from N. tomentosiformis. DNA sequence analysis reveals thatthe genomic fragments of NtabCYP82E5v2 and NtomCYP82E5 contain a 1054 bpintron flanked by a 604 bp 5′ and a 585 bp 3′ coding region and differat 15 positions of which nine and six are located within the codingregion and intron, respectively.

Furthermore, the coding regions of ten randomly selected genomic clonesisolated from tobacco share 100% nucleotide identity with theNtabCYP82E5v2 cDNA suggesting that NtabCYP82E5v2 is the only CYP82E5variant located in DH98-325-6 tobacco.

NtabCYP82E5v2 is Expressed at Low Levels in the Green and Yellow Leaves

To investigate the catalytic role of CYP82E5 in planta, thetranscriptional profile of CYP82E5v2 in the green and senescing leavesof converter (DH98-325-6) and nonconverter (DH98-325-5) Burley,converter and nonconverter Flue-Cured (SC58) tobacco, and N.tomentosiformis was determined using quantitative real-time (qrt)-PCRanalysis.

TABLE 2 Absolute quantification of the CYP82E4v2 and CYP82E5v2 cDNAderived from the green and cured leaves of N. tomentosiformis anddifferent tobacco genotypes by quantitative real-time polymerase chainreaction analysis. CYP82E4v2 CYP82E5v2 Green Cured Green Cured Sample PgN. tomentosiformis 0.35c 17.94e 1.73d 0.18c (0.16) (2.57) (0.72) (0.09)DH98-325-5 0.0004a 0.007b 0.08c 0.26c (0.0001) 0.003) (0.01) (0.17)DH98-325-6 0.004b 26.09e 0.137c 0.311c (0.001) (3.50) (0.003) (0.109)SC58NC 0.0006a 0.005b 0.070c 0.067c (0.0001) (0.003) (0.012) (0.005)SC58C 0.0006a 1.81d 0.11c 0.083c *Means are for 1 leaf of 3 independentplants, 3 cDNA measurements per sample (n = 9), except SC58C where oneplant was used (n = 3). *Numbers followed by different letters aresignificantly different according to Fisher's protected LSD (0.05).

Low levels of NtabCYP82E5v2 express in the green or yellow leaves of alltobacco cultivars (see, Table 2). The expression of NtabCYP82E5v2significantly increased in the cured versus green leaves of converterand nonconverter Burley cultivars but remained unchanged in Flue-Curedtobacco. In the green leaves of N. tomentosiformis, expression ofNtomCYP82E5 was higher than in tobacco, but no difference was notedbetween the cured leaves of the two species (Table 2). To compare theexpression levels of CYP82E5v2 with that of CYP82E4v2, a previouslycharacterized nicotine demethylase gene whose transcription was shown tobe sharply upregulated in senescing leaves of N. tomentosiformis andconverter tobacco (Gavilano et al. (2007) J. Biol. Chem. 282: 249-256),the transcript accumulation of CYP82E4v2 was compared in the sameextracts used for quantifying the CYP82E5v2 mRNA. Transcription ofNtabCYP82E5v2 is significantly higher than that of NtabCYP82E4v2 in thegreen leaves of all tobacco cultivars, N. tomentosiformis and in thecured leaves of nonconverter tobacco plants. However, the trend isreversed in the cured leaves of converter tobacco and N. tomentosiformis(Table 2). Of the two nicotine demethylase genes characterized thus farCYP82E4v2 is the dominant factor in senescence-inducible nornicotineproduction in N. tomentosiformis and tobacco. In addition, the activityof NtabCYP82E5v2 is the key determinant of nicotine conversion in thegreen leaves of tobacco.

Example 2: Sequence Identity to Cytochrome P450 Gene Family Members

Even though CYP82E5v2 and CYP82E4v2 are both nicotine demethylases, theyhave less sequence homology to each other than CYP82E5v2 does toCYP82E3, for example.

TABLE 3 Amino acid sequence identity shared between the CYP82E4v2nicotine demethylase enzyme and the other CYP82E2 proteins that havebeen assayed for nicotine demethylase activity. CYP82E4v6 CYP82E4v1258-166 CYP82E3 CYP82E2v1 CYP82E2v2 CYP82E5v2 CYP82E4v2 99.6% 99.4% 94.8%94.2% 92.6% 92.6% 91.1% SEQ ID NO: 13 CYP82E5v2 and CYP82E4v2 are theonly proteins represented that have nicotine demethylase activity.

TABLE 4 Amino acid sequence identity shared between the CYP82E4v2nicotine demethylase enzyme and other CYP82E2 proteins. CYP82E4v2CYP82E3 CYP82E2 CYP82E5v2 91.1% 91.9% 91.3% SEQ ID NO: 2 CYP82E5v2 andCYP82E4v2 are the only proteins represented that have nicotinedemethylase activity.

TABLE 5 Expression profiles for the CYP82E2 gene family members thathave been assayed for nicotine demethylase activity. CYP82E4v2 CYP82E4v6CYP82E4v12 58-166 CYP82E3 CYP82E2v1 CYP82E2v2 CYP82E5v2 Extremely Low inNot Extremely Low In Extremely Not Low in Low in Green Leaf; ReportedLow In Both Low in Reported Green Leaf; Green Leaf; High in Both Greenand Green Leaf; Very Low in Extremely Ethylene- Green and Ethylene-Extremely Ethylene- High in Induced Ethylene- Induced High in InducedEthylene- Senescent Induced Senescent Ethylene- Senescent Induced LeafSenescent Leaves Induced Leaf Senescent Leaves Senescent Leaf LeafCYP82E5v2 and CYP82E4v2 are the only proteins represented that werepositive for nicotine demethylase activity.

Example 3: mutations in CYP82E4V2 AND CYP82E5V2

Obtaining EMS-derived variants of CYP82E4v2

To facilitate the introduction of random mutations into the tobaccogenome, thousands of seeds of the strong Converter Burley lineDH98-325-6 were treated with the chemical mutagene ethyl methanesulphonate (EMS). Approximately 4000 mutagenized plants (Mo generation)were grown in the field and allowed to self-pollinate. Several capsulesfrom each individual plant were combined to create discrete M₁ seedpopulations, each corresponding to an individual Mo plant. Genomic DNAswere isolated from young leaf tissue of a single greenhouse-grown M₁plant from each MI seed pool. To identify plants carrying mutations inthe CYP82E4v2 gene, specific primers were designed to independentlyamplify each of the gene's two exons. DNA sequence information wasobtained using 96-well high-throughput sequence analysis of theamplification products. The complete analysis of 96 individual plantsinvolved 4 separate 96-well sequencing reactions: forward and reversesequencing of the exon 1 amplification product, and forward and reversesequencing of the exon 2 PCR product.

Mutations were identified by conducting a multi-sequence alignment ofall 96 sequences for a given run and looking for deviations from thewild-type sequence. Any given mutation that occurred in a parental M₀plant would be expected to be segregating in 1:2:1 ratio in the M₁generation for the mutant versus wild-type alleles. M₁ plants that arehomozygous for an EMS-induced mutation are readily recognized aspolymorphisms deviating from the wild type sequence, verified in bothdirections. M₁ plants that are heterozygous for a mutation in CYP82E4v2would be expected to have the mutant and wild type alleles eachrepresenting 50% of the amplification product. Upon sequencing, thelocation of such a mutation would be expected to be annotated as an “N”,since the fluorescence reading at that site would be a mixture of twoalternative bases. The appearance of an “N” at the same nucleotidelocation using the complementary primers, combined with visualinspection of the corresponding chromatograms distinguishes plants thatare truly heterozygous for a mutation in CYP82E4v2, and differentiatethese true heterozygotes from artifactual sequence anomalies.

Mutations in CYP82E4v2

High-throughput sequence analysis of 672 independent MI plants resultedin the identification of eleven individuals possessing point mutationswithin the CYP82E4v2 gene (see, Table 6). Six of the eleven plantsidentified carrying CYP82E4v2 mutations are homozygous for the mutantallele (see, Table 6). All eleven mutations result in changes in thepredicted amino acid sequence of the encoded protein.

TABLE 6 M₁ Lines of EMS Treated DH98-325-6 Plants Possessing Mutationsin the CYP82E4v2 Gene. Nucleotide Amino Acid Zygosity of Mutant PlantLine Mutation* Change** Allele DH98-325-6 #101 C1372T P458S HomozygousDH98-325-6 #121 G1092T K364N Homozygous DH98-325-6 #164 C113T P38LHomozygous DH98-325-6 #321 G601A E201K Heterozygous DH98-325-6 #377G506A R169Q Heterozygous DH98-325-6 #569 G1375A G459R HeterozygousDH98-325-6 #506 G886A E296K Heterozygous DH98-325-6 #453 C1280T T427IHeterozygous DH98-325-6 #775 G986A W329Stop Homozygous DH98-325-6 #610G1126A V376M Homozygous DH98-325-6 #761 G511A D171N Homozygous*Nucleotide location is in reference to the ATG initiator codon of theCYP82E4v2 cDNA (see SEQ ID NO: 50). Nucleotide to the left of the numberrepresents the wild type residue, whereas the nucleotide on the rightindicates the mutant residue (e.g. C1372T means that the C residuenormally located at position 1372 was mutated to a T residue). **Aminoacid location is in reference to the initiator methionine of theCYP82E4v2 polypeptide (see SEQ ID NO: 13). Amino acid to the left of thenumber represents the wild type residue, whereas the amino acid on theright indicates the residue produced by the mutant allele (e.g. P458Smeans that the Proline residue normally located at position 458 of theprotein sequence has been changed to a Serine). “Stop” indicates theintroduction of a premature stop codon.

Plant #775 possesses a mutation at codon 329 which changes a tryptophancodon (TGG) into a premature stop codon (TAG). This mutation renders theencoded protein completely nonfunctional given that the essentialheme-binding domain and other highly conserved regions of the enzyme arelocated downstream of codon 329. Other plants contain mutationsaffecting highly conserved motifs shared by the vast majority of P450enzymes include: plant #101 (where a conserved Pro residue immediatelyadjacent to the heme- binding Cys amino acid is change to a Ser); plant#164 (Pro residue within the highly conserved Pro-rich “hinge” region ofthe protein changed to a Leu); and plant #569 (Gly residue that's partof the very highly conserved heme-binding motif changed to an Arg).

Alkaloid Analysis of CYP82E4v2 Variants

An alkaloid analysis was conducted on cured leaves of variantindividuals to assess the effect of the mutation on the metabolicconversion of nicotine to nornicotine. As shown in Table 7, threeindividuals (#101, #121 and #775) show conversion rates similar tononconverter control plants grown from seed lots that had been screenedto eliminate converter individuals (TN90 LC plants). The variantCYP82E4v2 enzymes produced in these plants are completely (or nearlycompletely) nonfunctional. Two plants (#164 and #761) display highconversion rates and have no detrimental effect on enzyme activity. Thisresult for plant #164 is surprising given that it contains a Pro to Leumutation within one of the residues defining the highly conservedPro-rich hinge motif The Val to Met modification at residue 376partially inhibits enzyme function as the homozygous mutant plant (#610)shows an intermediate conversion phenotype (41.5%).

TABLE 7 Alkaloid Content as Percent Dry Weight of M₁ Plants Homozygousfor Mutations in the CYP82E4v2 Gene % Plant Nicotine NornicotineAnabasine Anatabine Conversion* DH98-325-6 #101 2.17 0.0376 0.005140.0384 1.7% DH98-325-6 #121 2.5 0.0333 0.00557 0.043 1.3% DH98-325-6#164 0.216 0.84 0.00499 0.0532 79.5% DH98-325-6 #610 0.214 0.152 0.004290.0121 41.5% DH98-325-6 #761 0.214 0.999 0.00429 0.0426 82.4% DH98-325-6#775 0.505 0.0159 0.00417 0.0136 3.1% SC58 CtCt (Control)^(†) 0.2060.565 0.00412 0.0197 73.3% TN90 LC (Control) 3.11 0.0647 0.00901 0.04872.0% TN90 LC (Control) 2.46 0.0521 0.00846 0.0417 2.1% *[%nornicotine/(% nicotine + % nornicotine)] × 100 ^(†)A converter plant offlue-cured genotype SC58 is used as a strong converter control; twoplants of genotype TN90 selected from lots screened for low converters(LC) are used as nonconverter controls.

The substitution of the Lys residue at position 364 to an Asn of plant#121 within this plant does not occur in any motif conserved among P450enzymes, nor within any region predicted by Xu et al. (PhysiologiaPlantarum 129: 307-319, 2007) to be a substrate recognition site. ABLAST alignment against protein sequences deposited in GenBank revealsseveral P450s that possess an Asn residue at the analagous position(e.g. the CYP82A1 gene of Pea—accession number Q43068). The variantCYP82E4v2 gene in plant #121 is completely inactive. Another anomalywith plant #121 lies in the nature of EMS-induced mutations. EMS isknown to induce mutations through the alkylation of G residues,resulting in G to A or C to T transition mutations (Anderson, MethodsCell Biol. 48:31-58, 1995). Very rarely does EMS lead to a G to Ttransversion mutation such as that found in plant #121 (see, Table 3).All other mutations described for either the CYP82E4v2 or CYP82E5v2genes are the expected G to A or C to T transition mutations.

Mutations in CYP82E5v2

A similar high-throughput sequencing strategy was used to identifymutations in CYP82E5v2, the other confirmed nicotine N-demethylase genethat is found within the tobacco genome. A screen of 768 M₁ plantsrevealed 11 individuals possessing mutations in CYP82E5v2 (see, Table8). Three plants (#744, #561 and #340) contain silent nucleotidesubstitutions that did not alter the predicted protein sequence. Of theeight mutations that lead to changes in the protein sequence, two are ofparticular note. Plant #1013 contains a mutation that would lead to atruncated product that should render the product completelynonfunctional. The G to A mutation at position 1266 changes a Trp codon(TGG) into a premature stop codon (TGA). The predicted CYP82E5v2 proteinproduced from this truncated reading frame lacks the final 96 aminoacids of the enzyme, a region that includes the essential heme-bindingdomain. Without being limited by mechanism, this truncated protein isprobably incapable of catalyzing the oxidative elimination of the N′methyl group of nicotine to form nornicotine. Whatever the mechanism,the #1013 plant has a nonfunctional CYP82E5v2 allele. Plant #680possesses a mutation that changes a Pro residue at position 449 to aLeu. This Pro is well conserved in plant P450s and lies immediatelyadjacent to the F-x-x-G-x-R-x-C-x-G motif that defines the heme bindingregion. The mutation found in plant #680 has a negative impact on enzymefunction.

TABLE 8 M₁ Lines of EMS Treated DH98-325-6 Plants Possessing Mutationsin the CYP82E5v2 Gene Nucleotide Amino Acid Zygosity of Mutant PlantLine Mutation* Change** Allele DH98-325-6 #198 C703T P235S HeterozygousDH98-325-6 #680 C1346T P449L Homozygous DH98-325-6 #744 G558A NoneHomozygous DH98-325-6 #163 C521T S174L Heterozygous DH98-325-6 #561G1458A None Heterozygous DH98-325-6 #154 C1229T A410V HeterozygousDH98-325-6 #340 G1248A None Heterozygous DH98-325-6 #780 G672A M224IHeterozygous DH98-325-6 #799 C215T P72L Heterozygous DH98-325-6 #540C427T L143F Heterozygous DH98-325-6 #1013 G1266A W422Stop Homozygous*Nucleotide location is in reference to the ATG initiator codon of theCYP82E5v2 cDNA (see SEQ ID NO: 1). Nucleotide to the left of the numberrepresents the wild type residue, whereas the nucleotide on the rightindicates the mutant residue (e.g. C703T means that the C residuenormally located at position 703 was mutated to a T residue). **Aminoacid location is in reference to the initiator methionine of theCYP82E5v2 polypeptide (see SEQ ID NO: 2). Amino acid to the left of thenumber represents the wild type residue, whereas the amino acid on theright indicates the residue produced by the mutant allele (e.g. P235Smeans that the Proline residue normally located at position 235 of theprotein sequence has been changed to a Serine). “Stop” indicates theintroduction of a premature stop codon. “None” indicates mutationsresulting in silent substitutions that did not alter the predicted aminoacid sequence.Plants with Mutations in CYP82E4v2 and CYP82E5v2

Tobacco plant lines are generated that show uniformity and stabilitywithin the limits of environmental influence for reduced conversion ofnicotine to nornicotine at levels of about 0.5% when a plant with amutation that inhibits nicotine demethylase activity of CYP82E4v2 iscrossed with a plant having a mutation that inhibits nicotinedemethylase activity of CYP82E5v2.

An illustrative cross is between a CYP82E4v2 mutant plant (of Table 9)and a CYP82E5v2 mutant plant (of Table 9).

TABLE 9 Illustrative Crosses of DH98-325-6 Plants Possessing Mutationsin the CYP82E5v2 and CYP82E4v2 Genes CYP82E5v2 DH98-325-6 #198DH98-325-6 #680 DH98-325-6 #163 DH98-325-6 #154 DH98-325-6 #780DH98-325-6 #799 DH98-325-6 #540 DH98-325-6 #1013 CYP82E4v2 DH98-325-6#101 DH98-325-6 #121 DH98-325-6 #775

All publications and patent applications mentioned in the specificationare indicative of the level of those skilled in the art to which thisinvention pertains. All publications and patent applications are hereinincorporated by reference to the same extent as if each individualpublication or patent application was specifically and individuallyindicated to be incorporated by reference.

Although the foregoing invention has been described in some detail byway of illustration and example for purposes of clarity ofunderstanding, it will be obvious that certain changes and modificationsmay be practiced within the scope of the appended claims.

1-31. (canceled)
 32. A tobacco seed, a tobacco plant grown therefrom, ora part of said tobacco plant, wherein said tobacco seed, tobacco plantgrown therefrom, or part of said tobacco plant comprises a variantpolynucleotide comprising a non-natural mutation as compared to areference sequence, wherein said reference sequence comprises at least95% sequence identity compared to SEQ ID NO:4, and wherein saidnon-natural mutation is positioned between two sequences correspondingto SEQ ID NOs: 36 and
 37. 33. The tobacco seed, a tobacco plant growntherefrom, or a part of said tobacco plant, according to claim 32,wherein said non-natural mutation is selected from the group consistingof a substitution, an addition, and a deletion of at least onenucleotide.
 34. The tobacco seed, a tobacco plant grown therefrom, or apart of said tobacco plant, according to claim 33, wherein saidnon-natural mutation is a null mutation.
 35. The tobacco seed, a tobaccoplant grown therefrom, or a part of said tobacco plant, according toclaim 33, wherein said tobacco plant is homozygous for said non-naturalmutation.
 36. The tobacco seed, a tobacco plant grown therefrom, or apart of said tobacco plant, according to claim 32, wherein saidpolynucleotide encodes a polypeptide that is not biologically active.37. The tobacco seed, a tobacco plant grown therefrom, or a part of saidtobacco plant, according to claim 32, wherein said tobacco plantcomprises a lower level of nornicotine or N′-nitrosonornicotine comparedto a tobacco plant comprising a wild type allele.
 38. The tobacco seed,a tobacco plant grown therefrom, or a part of said tobacco plant,according to claim 32, wherein the conversion of nicotine to nornicotinein tobacco material cured from said tobacco plant is selected from thegroup consisting of less than 0.3%, less than 0.5%, less than 0.7%,between 0.1%-0.5%, between 0.1%-0.4%, between 0.1%-0.7%, and between0.1%-1.0%.
 39. The tobacco seed, a tobacco plant grown therefrom, or apart of said tobacco plant, according to claim 32, wherein said tobaccoplant has a non-converter phenotype.
 40. The tobacco seed, a tobaccoplant grown therefrom, or a part of said tobacco plant, according toclaim 32, wherein said tobacco plant is selected from the groupconsisting of a Burley type, a dark type, a flue-cured type, and anOriental type.
 41. The tobacco seed, a tobacco plant grown therefrom, ora part of said tobacco plant, according to claim 32, wherein said seed,plant, or part further comprises a second variant polynucleotidecomprising a second non-natural mutation as compared to a secondreference sequence, wherein said second reference sequence comprises atleast 95% sequence identity compared to SEQ ID NO:13.
 42. A tobaccoseed, a tobacco plant grown therefrom, or a part of said tobacco plant,wherein said tobacco seed, tobacco plant grown therefrom, or part ofsaid tobacco plant comprises a variant polynucleotide comprising anon-natural mutation in a conserved residue of a polypeptide encoded bya reference sequence wherein said reference sequence comprises at least95% sequence identity compared to SEQ ID NO:4, and wherein saidconserved residue is a conserved residue as shown in FIG. 1A, 1B, or 1C.43. The tobacco seed, a tobacco plant grown therefrom, or a part of saidtobacco plant, according to claim 42, wherein said non-natural mutationis selected from the group consisting of a substitution, an addition,and a deletion of at least one nucleotide.
 44. The tobacco seed, atobacco plant grown therefrom, or a part of said tobacco plant,according to claim 43, wherein said non-natural mutation is a nullmutation.
 45. The tobacco seed, a tobacco plant grown therefrom, or apart of said tobacco plant, according to claim 43, wherein said tobaccoplant is homozygous for said non-natural mutation.
 46. The tobacco seed,a tobacco plant grown therefrom, or a part of said tobacco plant,according to claim 42, wherein said polynucleotide encodes a polypeptidethat is not biologically active.
 47. The tobacco seed, a tobacco plantgrown therefrom, or a part of said tobacco plant, according to claim 42,wherein said tobacco plant comprises a lower level of nornicotine orN′-nitrosonornicotine compared to a tobacco plant comprising a wild typeallele.
 48. The tobacco seed, a tobacco plant grown therefrom, or a partof said tobacco plant, according to claim 42, wherein the conversion ofnicotine to nornicotine in tobacco material cured from said tobaccoplant is selected from the group consisting of less than 0.3%, less than0.5%, less than 0.7%, between 0.1%-0.5%, between 0.1%-0.4%, between0.1%-0.7%, and between 0.1%-1.0%.
 49. The tobacco seed, a tobacco plantgrown therefrom, or a part of said tobacco plant, according to claim 42,wherein said tobacco plant has a non-converter phenotype.
 50. Thetobacco seed, a tobacco plant grown therefrom, or a part of said tobaccoplant, according to claim 42, wherein said seed, plant, or part furthercomprises a second variant polynucleotide comprising a secondnon-natural mutation as compared to a second reference sequence, whereinsaid second reference sequence comprises at least 95% sequence identitycompared to SEQ ID NO:13.
 51. The tobacco seed, a tobacco plant growntherefrom, or a part of said tobacco plant, according to claim 42,wherein said tobacco plant is selected from the group consisting of aBurley type, a dark type, a flue-cured type, and an Oriental type.